WO2008012932A1 - Insulin resistance reducer - Google Patents

Abstract

As one example of investigating effects of accumulation of chondroitin sulfate proteoglycan (CSPG), the invention relates to a versican siRNA containing a core site sequence of versican which is one of the chondroitin sulfate proteoglycans, an siRNA of C4ST1, C4ST2 or C4ST3 which is an enzyme transferring a sulfate group of acetylgalactosamine being a side chain of chondroitin sulfate proteoglycan, and an insulin resistance reducer suitable for gene therapy or prevention for a disease caused by an increase in insulin resistance such as type II diabetes (NIDDM). The invention also relates to a method of reducing insulin resistance based on inhibiting the accumulation of chondroitin sulfate proteoglycan (CSPG) by administering a versican siRNA, C4ST-1 siRNA, C4ST-2 siRNA or C4ST-3 siRNA to a test subject and a method of treating or preventing a disease caused by an increase in insulin resistance such as type II diabetes utilizing the same.

Description

 Insulin resistance inhibitor

 Technical field

 [0001] The present invention relates to an insulin resistance inhibitor based on control of accumulation of chondroitin sulfate proteoglycan (CSPG), a method for suppressing insulin resistance, and insulin-independent type 2 diabetes based on the method The present invention relates to a method for treating or preventing insulin resistance diseases such as diseases (NIDDM).

 Background art

 [0002] Diabetes is a disease caused by a relative shortage of insulin, a hormone that lowers blood glucose, and symptoms vary from person to person. It is highly insulin dependent type 1 (IDDM) and insulin independent. Can be divided into type 2 (NIDDM). Type 1 diabetes (ID DM) is a condition in which the splenic j8 cells that inherently produce insulin are destroyed by autoimmunity. On the other hand, type 2 diabetes mellitus (NIDDM) is a well-known lifestyle-related disease that has developed due to the addition of environmental factors such as obesity and lack of exercise to genetic factors. Current diabetes symptomatic treatment and pharmacotherapy are sufficient diets that keep nutrients (sugars, lipids, proteins) lances and do not run out of minerals and vitamins, 10 minutes or longer, 30 if possible Exercise therapy with continuous whole body exercise for more than min., Nateglide, a drug targeting insulin-related enzymes and insulin receptors as drug therapy, a-darcosidase inhibitor, sulfo-urea agent (SU agent), Oral diabetes drugs such as biguanides (BG) and thiazolidine drugs are used in clinical settings. (See Non-Patent Documents 1-5)

[0003] However, it has not been able to improve the patient's QOL and effectively prevent or delay the decrease in insulin secretion. However, according to a recent research report by the Ministry of Health and Welfare, it is not dependent on insulin. The prevalence of diabetes mellitus (NIDDM) is about 10% of the population over the age of 40, reaching about 6 million people in Japan as a whole. Changes in society and an aging society are accelerating the increase in diabetes and are continuing to increase, and safety is completely different from the past. There is an urgent need for the development of new drugs that are superior in their ability to effectively prevent or delay.

 [0004] Currently, diabetes is being studied all over the world as a target after cancer. In the midst of various drug discovery, recently, “regeneration of spleen ι8 cells” has been expected as one of the fundamental therapies. (See Non-Patent Documents 6-12)

 [0005] A proteodalycan is a molecule having a structure in which one or more glycosaminoglycan (GAG) chains are covalently bonded to a protein called a core protein, and the specific sugar chain structure of the GAG chain is a proteoglycan. Proteodarican is based on the type of GAG chain, and chondroitin sulfate proteodarican, dermatan sulfate proteodarican, henoran sulfate proteodarican, and ketalan sulfate proteodarican. Broadly divided into four. (See Non-Patent Documents 13-19)

 [0006] Among these, heparan sulfate proteodaricans have been extensively studied to bind to various site force-ins, adhesion molecules, and chemokines and greatly modify their functions.

 [0007] On the other hand, chondroitin sulfate proteodarican is an essential molecule during the embryonic period and is abundant in each organ, but is known to decrease with birth, growth, and aging. Kang's in vivo function has not yet become clear. To date, chondroitin sulfate proteoglycan (CSPG), heparan sulfate proteoglycan (HSPG), dermatan sulfate proteoglycan (DSPG), basic fibroblast growtn factor (bFu) ^ decorm, biglycan, lumican It has been reported to be expressed and accumulated in glomerular basement membrane, spleen, kidney and liver of mice and rats. (See Non-Patent Documents 20-26)

 [0008] However, the significance of CSPG increase in these diseases has not been clarified at all, and in recent years it has been found that mice genetically deficient in CSPG fall into embryonic lethality and organ dysplasia. The recognition that CSPG is an indispensable molecule for living organisms is increasing.

[0009] Versican (aka PG-M), a chondroitin sulfate proteodarican, has a hyaluronic acid binding domain near the N-terminal, a glycosaminodarlican addition domain in the middle, an EGF-like domain in the C-terminal, C It has a type lectin-like domain and a complement regulatory protein-like domain. The human Versican gene is composed of 15 exons, and as a result of alternative splicing, Ve rsican takes four different molecular types: V0, VI, V2 with different glycosaminodalican-capped domain lengths, and V3 without glycosaminoglycan-capped domain. Versican binds hyaluronic acid with high affinity via the hyaluronic acid-binding domain, and binds to sulfated glycolipids and extracellular matrix components tenascin-Ryafibrin-1 via the C-type lectin domain. In addition, Versican has been reported to promote cell proliferation at least partially by binding to the EGF receptor via the EGF-like domain, and also reported to have cell adhesion inhibitory activity via the chondroitin sulfate chain. Speak. (See Non-Patent Documents 27-33)

 [0010] Currently, sulfo-urea drugs (SU agents), biguanide drugs, insulin resistance improvers, anti-darcosidase inhibitors, fast-acting insulin secretagogues, etc. are used as antidiabetic drugs. However, research is still ongoing as a pathological field that is difficult to treat.

 [0011] To date, Versican is one of the chondroitin sulfate proteoglycans (CSPG) and is known as a protein corresponding to the core protein. Regarding chondroitin sulfate proteodarican, neural stem cell differentiation inducer (Patent Document 1), nerve regeneration using human Z bone morphogenetic protein (Patent Document 2), glycosyl transfer agent (Patent Document 3), central nervous system damage (Patent document 4), nerve regeneration using human, bone morphogenetic protein (patent document 5), therapeutic inhibitor of vascular smooth muscle cells (patent document 6), etc. Darican growth promoter (Patent Literature 7), hair growth composition (Patent Literature 8), ADAMTS4 function inhibitor (Patent Literature 9), and a method for suppressing scar tissue formation using an implantable medical device (Patent Literature) 10) etc. are known.

 [0012] Also, in US PATENT, PAT.NO.5,180,808 (patent document 11) related to structure, nucleic acid base sequence related, etc. and PAT.NO.6,579,682 (patent document 12) related to cardiovascular diseases Patents have been filed, and PubMedline reports on structures, cardiovascular systems, and reports on Versican and metabolic disorders are reported in a paper on lipid metabolism (Non-patent Document 34). Not limited to Versican siRNA, C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA administration examples and efficacy studies using the mouse type 2 diabetes (NIDDM) model. .

 [0013] Patent Document 1: Patent Publication 2005- 278641

Patent Document 2: Patent Publication 2005-007196 Patent Document 3: Patent Publication 2004-024208

Patent Document 4: Patent Publication 2005-526740

Patent Document 5: Patent Publication No. 09-501932

Patent Document 6: Patent Publication 08-510209

Patent Document 7: Patent Publication 2006-028071

Patent Document 8: Patent Publication 2005-200383

Patent Document 9: Patent Publication 2004-244339

Patent Literature 10: Patent Publication No. 07-024053

Patent Document 11: US Pat. No. 5,180,808

Patent Document 12: US Pat. No. 6,579,682

Non-patent literature l: Fujita, T et al. Biochemical Pharmacology (1996); 52: 407-411 Non-patent literature 2: Tsukuda, K et al. Horm Metab Res (1998); 30: 42-49

Non-Patent Document 3: Fujitani, S et al. Metabolism (1996); 45: 184-189

Non-Patent Document 4: Spiegelman et al. J Clin Invest (1997); 100: 1863-1869

Non-Patent Document 5: 01efsky et al. Diabetes (1997); 46: 1678-1683

Non-patent literature 6: Glueck CJ et al. Curr Diab Rep. (2003) Aug; 3 (4): 303-312 Non-patent literature 7: Juntunen KS et al. Am J ClinNutr. (2003) Feb; 77 (2) : 385-391 Non-Patent Document 8: Van Lerberghe S et al. Diabeets Metab. (2002) Feb; 28 (1): 33-38 Non-Patent Document 9: Buchanan TA. J Clin Endocrinol Metab. (2001) Mar; 86 (3): 989-993 Non-Patent Document 10: Fallucca F et al. Diabetes Res Clin Pract. (1989) Nov 6; 7 (4): 277-2

84

Non Patent Literature l l: Ostenson CG et al. Exp Clin Endocrinol. (1989) May; 93 (2-3): 241- 247

Non-Patent Document 12: Ward WK et al. J Clin Endocrinol Metab. (1985) Dec; 61 (6): 1039-10 45

Non-Patent Document 13: Lindahl, U et al. (1972) In Glycoproteins (Gottschalk, A. ed) pp. 491 —517, Elsevier, New York.

Non-Patent Document 14: Oegema, T et al. J. Biol. Chem. (1984); 259: 1720-1726 Non-patent literature 15: Sugahara, K et al. J. Biol. Chem. (1988); 263: 10168-10174 Non-patent literature 16: Sugahara, K et al. J. Biol. Chem. (1992); 267: 6027 -6035 Non-Patent Document 17: De Waard et al. J. Biol. Chem. (1992); 267: 6036-6043

Non-Patent Document 18: Moses, J, Oldberg et al. Eur. J. Biol. (1992); 248: 521-526 Non-Patent Document 19: Yamada, S et al. Trends in Glycoscience and Glycotechnology,. (199

8); 10: 95-123

Non-Patent Document 20: Schaefer L et al. FASEB J. (2001) Mar; 15 (3): 559-561

Non-Patent Document 21: McCarthy K.J et al. J Histochem Cytochem. (1994) Apr; 42 (4): 473-48

Four

Non-Patent Document 22: Hanneken A et al. Arch Ophthalmol. (1991) Jul; 109 (7): 1005-1011 Non-Patent Document 23: Fushimi H et al. J Intern Med. (1989) Dec; 226 (6): 409-416 Non-Patent Document 24: Klein DJ et al Diabetes. (1989) Jan; 38 (1): 130-139

Non-Patent Document 25: Klein D.J et al. Diabetes. (1986) Oct; 35 (10): 1130-1142

Non-Patent Document 26: Rohrbach DH et al. J Biol Chem. (1983) Oct 10; 258 (19): 11672-1167

7

Non-Patent Document 27: Krusius T et al. J Biol Chem. (1987) Sep 25; 262 (27): 13120-13125 Non-Patent Document 28: Naso MF et al. J Biol Chem. (1994) Dec 30; 269 ( 52): 32999-33008 Non-patent document 29: Ito K et al. J Biol Chem. (1995) Jan 13; 270 (2): 958-965

Non-Patent Document 30: Shinomura T et al. J Biol Chem. (1995) Apr 28; 270 (17): 10328-103

33

Non-patent document 31: Zako M et al. J Biol Chem. (1995) Feb 24; 270 (8): 3914-3918 Non-patent document 32: Yang BL et al. J Cell Biochem. (1999) Feb 1; 72 ( 2): 210-220 Non-patent literature 33: Aspberg A et al. J Biol Chem. (1999) Jul 16; 274 (29): 20444-20449 Non-patent literature 34: Davidsson P et al. J Lipid Res. (2005 ) Sep; 46 (9): 1999-2006 Disclosure of the Invention

Problems to be solved by the invention

The present invention imposes provision of an insulin resistance inhibitor, a therapeutic agent for an insulin resistance disease containing the drug as an active ingredient, and a method for screening an insulin resistance inhibitor. The title.

 Means for solving the problem

 [0015] The present invention relates to an agent capable of suppressing chondroitin sulfate proteodarican (CSPG) accumulation in spleen β cells, useful for the treatment or prevention of non-insulin dependent diabetes mellitus (NIDDM) based on chondroitin sulfate proteodarican (CSPG) accumulation One objective is to provide

 [0016] The present inventors have conducted intensive research to develop such a drug, and have been considered to be the etiology of insulin-resistant diseases so far, and the strong chondroitin sulfate proteodarican (C SPG) Excessive accumulation may promote insulin resistance of spleen β-cells

[0017] As a result of conducting research based on this idea, the present inventors have found that Versican, which is one of chondroitin sulfate proteoglycans (CSPG), and C4ST-, which is one of chondroitin sulfate proteoglycans (CSPG), sulfotransferases. 1. Treating C4ST-2 and C4ST-3 genes with siRNA suppresses CSPG expression in spleen β-cells, increases insulin-positive β-cell expression, decreases expression of amyloid precursor protein, and significantly increases insulin resistance I found out that it can be suppressed. That is, it was demonstrated that insulin resistance can be suppressed by inhibiting the accumulation or biosynthesis of chondroitin sulfate proteodarican, and the present invention was completed. Substances that inhibit the production or accumulation of chondroitin sulfate proteodalycan are useful as insulin resistance inhibitors. In addition, the drug is a drug for treating or preventing an insulin resistant disease.

 [0018] The present invention relates to an insulin resistance inhibitor, a therapeutic agent for an insulin resistance disease containing the drug as an active ingredient, a screening method for an insulin resistance inhibitor, and the like, more specifically,

 [1] an insulin resistance inhibitor comprising as an active ingredient a substance that inhibits the production or accumulation of chondroitin sulfate proteodarican,

 [2] The drug according to [1], wherein the substance is a substance having a chondroitin sulfate proteodarican degradation promoting action,

[3] A substance, wherein the substance has a chondroitin sulfate proteodarican synthesis inhibitory action The drug according to [1],

 [4] The drug according to [1], wherein the substance is a substance having a chondroitin sulfate proteodarican desulfation action,

 [5] The drug according to [1], wherein the substance is a substance having an inhibitory effect on chondroitin sulfate proteodalycan sulfate,

 [6] The drug according to any one of [1] to [5], wherein production or accumulation of chondroitin sulfate proteodalycan is inhibited in spleen β cells,

[7] The drug according to any one of [1] to [6] for treatment or prevention of insulin resistance disease,

 [8] The drug according to [7], wherein the insulin resistance disease is an insulin resistance disease associated with metabolic syndrome,

 [9] the insulin-resistant disease force S insulin-independent type (type 2) diabetes, the drug according to [7],

 [10] A screening method for an insulin resistance inhibitor, which comprises selecting a substance having an action of inhibiting the production or accumulation of chondroitin sulfate proteodarican from a test sample,

 [11] The screening method according to [10], comprising a step of selecting a substance having the action described in any of (a) to (d) below,

( _a ) Promoting the degradation of chondroitin sulfate proteodarican

 (b) Inhibition of chondroitin sulfate proteodarican synthesis

 (c) Desulfation effect of chondroitin sulfate proteodarican

 (d) Sulfate inhibitory action of chondroitin sulfate proteodarican

 [12] The screening method according to [10] or [11], wherein the insulin resistance inhibitor is for treatment or prevention of an insulin resistance disease,

Is provided.

 The present invention further relates to the following.

[13] Use of the drug according to any one of [1] to [9] in the manufacture of an insulin resistance inhibitor, [14] A method for treating an insulin resistant disease, comprising a step of administering the drug according to any one of [1] to [9] to an individual (patient etc.),

 [15] A composition comprising the drug according to any one of [1] to [9] and a pharmaceutically acceptable carrier.

 The invention's effect

 [0019] The present invention has revealed that the development and accumulation of chondroitin sulfate proteodalycan is related to the development of insulin resistance. It was shown that insulin resistance is suppressed by inhibiting the production and accumulation of chondroitin sulfate proteoglycan. It will be possible to provide a new concept of therapeutic drugs for insulin resistance. Insulin resistance in particular is closely related to metabolic syndrome (visceral fat syndrome) such as diabetes 'hyperlipidemia, obesity' arteriosclerosis, and the number of patients is increasing in modern society. It has significant medical and industrial significance.

 Brief Description of Drawings

 [0020] [Fig. L] Diagram showing changes in body weight during 14 days in a control group and a Versican siRNA-treated group in a type 2 diabetes (NIDDM) model mouse (female) induced by Streptozotocin. is there. The vertical axis represents weight (g) and the horizontal axis represents days.

 [Fig.2] Versican siRNA-treated model in type 2 diabetes (NIDDM) model mice (female) induced by Streptozotocin! After insulin tolerance test, 0 min, 15 min, It is a figure which shows the fluctuation | variation of the blood glucose level in a Control (control) group 60 minutes later, and a Versican (Versican) siRNA treatment group. The vertical axis represents the blood glucose level (mg / dL), and the horizontal axis represents the blood glucose measurement time (min) after the insulin load test. * p <0.05, ** p <0.01 (t-test)

 [Fig. 3] GAPDH, Versican (Versican) by RT-PCR on the 14th day (last day) in the control group, Versican (Versican) siRNA treatment group in type 2 diabetes (NIDDM) model mice induced by Streptozotocin ). Versican (Versican) expression is suppressed in the siRNA-treated group!

[Figure 4] Reduced deposition of Amyloid precursor protein (APP) in Versi can siRNA-treated mice in type 2 diabetes (NIDDM) model mice induced by Streptozotocin ( (Upper), a photograph showing detection results (red brown signal, blue signal) of insulin positive spleen β-cell rise (lower). Magnification 500 times (upper), 200 times (lower).

 [Figure 5] Detection of chondroitin sulfate proteodarican (CSPG) in the control group, Versican siRNA treated group in type 2 diabetes (NIDDM) model mice induced by Streptozotocin Versican siRNA It is a photograph showing the reduction effect of chondroitin sulfate proteodarican (CSPG) (purple signal) in the treatment group. 400x magnification. The name of the antibody clone used for detection is shown below the photograph.

 [Figure 6] 14-day body weight changes in the control group, C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA treatment group in type 2 diabetes (NIDDM) model mice induced by Streptozotocin FIG. The vertical axis represents body weight (g), and the horizontal axis represents days.

 [Fig. 7] Streptozotocin-induced type 2 diabetes mellitus (NIDDM) model mice treated with C 4ST-1 siRNA, C4ST-2 siRNA, C4ST-3 siRNA treated model after 0 min, 15 min after insulin load test FIG. 6 is a graph showing changes in blood glucose levels in the Control group, C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA treatment group after 60 minutes. The vertical axis represents blood glucose level (mg / dL), and the horizontal axis represents blood glucose measurement time (min) after the insulin tolerance test. * p <0.05, ** p <0.01 (t-test)

 [Fig. 8] Day 14 (last day) in the control group, C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA treatment group in type 2 diabetes (NIDDM) model mice induced by Streptozotocin FIG. 5 shows the expression of GAPDH and Versican by RT-PCR. C4ST-1 siRNA, C4ST-2 siRNA, C4ST-3 This shows that C4ST-1, C4ST-2, and C4ST-3 expression was suppressed in the siRNA treatment group! /.

 [Fig. 9] C4ST in type 2 diabetes (NIDDM) model mice induced by Streptozotocin

-1 Photographs showing detection results (red-brown signal, blue signal) of decreased Amyloid precursor protein (APP) deposition (upper) and elevated insulin-positive spleen β-cells (lower) in siRNA-treated groups. Magnification ₅₀₀ times (upper), ₂₀₀ times (lower).

[Fig. 10] Chondroitin sulfate protease in control group and C4ST-1 siRNA treatment group in type 2 diabetes (NIDDM) model mice induced by Streptozotocin It is a photograph showing the reduction effect of chondroitin sulfate proteoglycan (CSPG) (purple signal) in the C4ST-1 siRNA treatment group as a result of detection of oglycan (CSPG). 400x magnification. The name of the antibody clone used for detection is shown below the photo.

 BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in detail.

 The pathological conditions associated with diabetes, one of the typical metabolic syndromes (visceral fat syndrome), include abnormal insulin secretion in splenic j8 cells and impaired insulin action, including glucose uptake in insulin target tissues. The present inventors have paid attention to the function of chondroitin sulfate proteodalycan in order to improve the abnormal secretion of insulin in splenic j8 cells as one of the effective methods for treating diabetes. Then, a state in which accumulation of chondroitin sulfate proteodarican was suppressed in a diabetic model mouse was created and analyzed in detail. As a result, the accumulation of chondroitin sulfate proteodarican was improved compared to wild-type spleen j8 cells. Many of the cells were observed, and the condition of diabetes such as blood glucose level was improved. In other words, inhibiting the production or accumulation of chondroitin sulfate proteodarican promotes improvement of abnormal accumulation state of chondroitin sulfate proteodarican in splenic j8 cells that are deeply involved in diabetes, leading to improvement of insulin resistance. I found.

 [0022] The present invention relates to an insulin resistance inhibitor comprising as an active ingredient a substance that inhibits the production or accumulation of chondroitin sulfate proteodalycan.

[0023] The "chondroitin sulfate proteodarican" of the present invention is one of the proteodaricans, and is a covalent bond between chondroitin sulfate Z dermatan sulfate, which is a typical sulfated mucopolysaccharide, and protein (coprotein). A generic term for compounds. The “chondroitin sulfate proteodarican” in the present invention is preferably a human chondroitin sulfate proteodarican, but the species from which it is derived is not particularly limited. Proteins equivalent to can (such as homologs and orthologs) are also included in “chondroitin sulfate proteodaricans” in the present invention. For example, the present invention can be carried out as long as the organism has a protein corresponding to human chondroitin sulfate proteodalycan and has a protein equivalent to human chondroitin sulfate proteoglycan. In addition, the chondroitin sulfate proteodarican in the present invention is one of the causes of inflammation. Occasionally, glycosaminodarlican (GAG) chains are combined to become proteodaricans, so-called part-time proteodaricans.

[0024] In the following description, examples of chondroitin sulfate proteoglycans include aggrican, versican, neurocan, brevican, β-glycan, Decorm, Biglycan, Fibromodulin, and PG-Lb. The chondroitin sulfate proteodarican in the present invention is not limited to these, and any substance having activity as a chondroitin sulfate proteodarican can be used. Here, the activity of chondroitin sulfate proteodalycan includes, for example, cell adhesion ability or cell growth promotion. A person skilled in the art can evaluate the activity as chondroitin sulfate proteodalycan by the following method. A protein containing a partial region of chondroitin sulfate proteodarican amino acid sequence, or a high homology with a partial region (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably Measure the divisional proliferation of tumor cells (eg Caco-2, HT-29 cells, etc.) in the presence of proteins with greater than 95%). Proteins that have the effect of promoting mitotic proliferation can be determined as proteins with chondroitin sulfate proteodarican activity (Int J Exp Pathol. 2005 Aug; 86 (4): 219-29 and Histochem Cell Biol. 2005 Aug; 124 (2): 139-49). Here, high homology means 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more (e.g., 95% or more, further 96%, 97%, 98% or 99% or higher) homology. This homology is determined by the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 8 7: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873— 7).

 “Insulin resistance” in the present invention refers to a state in which the presence of insulin in a living body is abnormal. For example, the ability to reduce insulin secretion such as β-cell force in the spleen and the action of insulin in the target tissues of skeletal muscle, liver, and adipose tissue are not limited to these.

In the present invention, “inhibiting the production or accumulation of chondroitin sulfate proteodarican” means, for example, “promotion of degradation”, “synthesis inhibition”, “desulfation”, “sulfate sulfate” But not limited to them, the abundance, function or activity of chondroitin sulfate proteodarican is reduced or eliminated compared to the comparison target. That means. In the present invention, the “substance that inhibits the production or accumulation” of chondroitin sulfate proteodarican is not particularly limited, but preferably the “substance that has an activity of promoting degradation of chondroitin sulfate proteoglycan” and “the substance has an inhibitory effect on synthesis”. “Substance”, “Substance with desulfurization and oxidation”, or “Substance with sulfation-inhibiting action”.

 [0027] "Promoting degradation" of chondroitin sulfate proteodarican includes, for example, inhibition of the expression of the protein that is the core of chondroitin sulfate proteodarican. Here, the “protein that is the core of chondroitin sulfate proteodarican” includes, for example, core proteins such as aggrican, versican, neurocan, and b revican in the case of matri X type chondroitin sulfate proteoglycan. Examples of membrane-type chondroitin sulfate proteodlicans include core proteins such as j8 glycan, Decorin, Biglycan, Fibromodulin, and PG-Lb. These are only examples, and are not limited to these, and may be any protein that is widely used as the core of chondroitin sulfate proteodalycan.

 “Expression” includes “transcription” from a gene or “translation” into a polypeptide and “degradation inhibition” of a protein. “Expression of the protein that is the core of chondroitin sulfate proteodarican” refers to the transcription and translation of the gene that encodes the protein that is the core of chondroitin sulfate proteodarican, or the chondroitin sulfate proteo This means that the protein that forms the core of Darican is produced. In addition, “the function of the protein serving as the core of chondroitin sulfate proteodarican” includes, for example, the function of the protein binding to chondroitin sulfate and the binding to other components in the cell. The various functions described above can be appropriately evaluated (measured) by those skilled in the art using common techniques. Specifically, the methods described in the examples described later, or the methods can be appropriately modified and carried out.

[0029] Furthermore, "degradation promotion" of chondroitin sulfate proteodarican may be an increase in the expression of an enzyme that cleaves or degrades chondroitin sulfate proteodarican or an enzyme related thereto. Examples of these enzymes include, but are not limited to, meta-oral proteinases (for example, AD AMTS-1, ADAMTS-4, ADAMTS-5, etc.) chondroitinase, Calpain I, and the like. “Acceleration of degradation” is a decrease in the abundance of chondroitin sulfate proteodarican caused by administration of these enzymes or a part of them. A little.

 [0030] "Degradation promotion" may be caused by administration of a substance that promotes suppression of chondroitin sulfate proteodarican expression. These substances include, for example, n-butylate, Diethyl carbamazepine, i'unicamycin, non-steroidal estrogen, and cyclofenil deiphenol.

 [0031] Preferable embodiments of the "substance having a decomposition promoting action" include, for example, a compound (nucleic acid) selected from the group consisting of the following (a) to (c).

( _a ) An antisense nucleic acid for a transcription product of a gene encoding the core protein of chondroitin sulfate proteodarican or a part thereof

 (b) a nucleic acid having a ribozyme activity that specifically cleaves the transcription product of the gene encoding the core protein of chondroitin sulfate proteodarican

 (c) Nucleic acid that acts to inhibit the expression of the gene encoding the core protein of chondroitin sulfate proteodarican by the RNAi effect

 In addition, examples of the “substance having a decomposition promoting action” include compounds selected from the following groups (a) to (c).

( _a ) an antibody that binds to the core protein of chondroitin sulfate proteodarican

 (b) Chondroitin sulfate proteodarican mutants having dominant negative properties for the core protein of chondroitin sulfate proteodarican

 (c) Low molecular weight compound that binds to the core protein of chondroitin sulfate proteodarican [0033] “Synthetic inhibition” of chondroitin sulfate proteodarican means, for example, inhibition of glycosaminodarlican biosynthesis, chondroitin sulfate proteodarican synthesis Inhibition of enzymes involved in the above, but is not necessarily limited to these, it refers to inhibiting any of the processes in which chondroitin sulfate proteodarican is synthesized.

[0034] Examples of substances that inhibit the synthesis of chondroitin sulfate proteodarican include those that inhibit glycosaminoglycan biosynthesis, such as j8-D-xyloside, 2-deoxy-D-glucose (2-D (J), ethane-1-hydroxy-1, 1-diphosphonate (ETDP), 5-hexoxy 2-aeoxyundine (H UdR), etc. The biosynthesis of glycosaminodarlicans can be achieved with these and other substances. Inhibited and the synthesis of chondroitin sulfate proteodalycan is inhibited. On the other hand, examples of enzymes involved in chondroitin synthesis include GalNAc4ST-1, GalNAc4 ST-2, GALNAC4S-6ST, UA20ST, GalT-I, GalT-II, GlcAT-I, and XylosylT. By inhibiting these and other enzymes and suppressing their expression, the synthesis of chondroitin sulfate proteodalycan is inhibited.

 [0036] Preferable embodiments of the "substance having a synthesis inhibitory action" include, for example, a compound (nucleic acid) selected from the group consisting of the following (a) to (c).

( _a ) An antisense nucleic acid for a transcript or a part of a gene encoding chondroitin sulfate proteodarican synthase

 (b) Nucleic acid with ribozyme activity that specifically cleaves the transcript of the gene encoding chondroitin sulfate proteodarican synthase

 (c) a nucleic acid having an action of inhibiting the expression of a gene encoding chondroitin sulfate proteodarican synthase by the RNAi effect

 [0037] In addition, examples of the "substance having a synthesis inhibitory action" include compounds selected from the following groups (a) to (c).

( _a ) an antibody that binds to chondroitin sulfate proteodalycan synthase

 (b) Chondroitin sulfate proteodarican synthase mutant having dominant negative properties for chondroitin sulfate proteodarican synthase

 (c) Low molecular weight compound that binds to chondroitin sulfate proteodarican synthase

 [0038] "Desulfation" of chondroitin sulfate proteodarican refers to removal of sulfate groups in chondroitin sulfate proteodarican, for example, desulfation or sulfation by a desulfase enzyme that is administered with endogenous or external force. Examples include, but are not limited to, suppression of sulfation by a compound that suppresses sulfation.

 [0039] Examples of the desulfating enzyme include Chondroitin-4-sulfatase and Chondroitin-6-sulfatase. Examples of the compound that suppresses sulfation include Chlorate and EGF receptor antagonist.

 [0040] Preferable embodiments of the "substance having desulfating action" include, for example, compounds (nucleic acids) selected from the group consisting of the following (a) to (c).

( _a ) _a residue encoding a chondroitin sulfate proteodarican desulfation enzyme inhibitory protein Antisense nucleic acid for gene transcripts or parts thereof

 (b) a nucleic acid having a ribozyme activity that specifically cleaves a transcript of a gene encoding a chondroitin sulfate proteodarican desulfating enzyme inhibitory protein

 (C) a nucleic acid having an action of inhibiting the expression of a gene encoding a chondroitin sulfate proteodarican desulfating enzyme inhibitory protein by the RNAi effect

[0041] Examples of the "substance having a desulfating action" include compounds selected from the following groups (a) to (c).

( _a ) an antibody that binds to _a chondroitin sulfate proteodarican desulfase inhibitor compound

(b) Chondroitin sulfate proteodarican desulfate-enzyme inhibitory protein against chondroitin sulfate proteodalycan desulfate-enzyme inhibitory protein

 (c) a small molecule compound that binds to a chondroitin sulfate proteodarican desulfase inhibitor compound

 Here, the “desulfation-inhibiting compound” is not limited to a protein, and includes non-protein compounds such as coenzymes, for example.

[0043] The "sulfate inhibitory action" of chondroitin sulfate proteodarican includes, for example, inhibition of sulfate group transfer enzyme, but is not limited to this, and occurs in the process of chondroitin sulfate proteodarican synthesis. It refers to inhibition of sulfation.

[0044] Examples of the sulfotransferase include C4ST-l (Chondroitin D-N-acetylgalactosamine

-4-0-sulfotransferas e 1), 4ST-2 (Chondroitin D—N—acetylgalactosamine—4-0—sulfotransferase 2), C4ST-3 (Chondroitin DN-acetylgalactosamine-4-0-sulfotransferas e 3), D4ST C6ST-1, C6ST-2 and the like.

[0045] Preferable embodiments of the "substance having a sulfate inhibitory effect" include, for example, compounds (nucleic acids) selected from the following groups (a) to (c).

( _a ) An antisense nucleic acid for a transcript or a part of a gene encoding chondroitin sulfate proteodalycan sulfate transferase

(b) A nucleic acid having a ribozyme activity that specifically cleaves a transcript of a gene encoding chondroitin sulfate proteodarican sulfate transferase. (c) Nucleic acid having the action of inhibiting the expression of the gene encoding chondroitin sulfate proteodarican sulfate transferase by the RNAi effect

In addition, examples of the “substance having a sulfation inhibiting action” include compounds selected from the group consisting of the following (a) to (c).

( _a ) an antibody that binds to chondroitin sulfate proteodalycan sulfate transferase

 (b) Chondroitin sulfate proteodalycan sulfate transferase mutant

 (c) A low molecular weight compound that binds to chondroitin sulfate proteodalycan sulfate transferase

[0047] The enzymes exemplified above include not only one enzyme corresponding to one gene but also an enzyme group sharing certain characteristics. For example, chondroitinase is a collective term for enzymes such as ABC, AC, and B that share the characteristics of mucopolysaccharide-degrading enzymes but differ in substrate specificity. For example, chondroitinase AC I cleaves the chondroitin sulfates (A, C or E), chondroitin, chondroitin sulfate-dermatan sulfate hybrid type and hyaluronic acid N-acetylhexoxide binding bond. Thus, an oligosaccharide having a Δ 4-glucuronic acid residue at the non-reducing end is generated. This enzyme does not act on dermatan sulfate (chondroitin sulfate B, which has L-iduronic acid as hexuronic acid), ketalan sulfate, heparan sulfate and heparin. In addition, chondroitinase AC II cleaves the N-acetyl hexosaminide bond of chondroitin, chondroitin sulfate A and chondroitin sulfate C in an elimination reaction, resulting in 厶 4-unsaturated disaccharide (0 卜 03, AD 4S and A Di-6S). This enzyme also works well on hyaluronic acid. It does not act on dermatan sulfate (chondroitin sulfate B) and is a competitive inhibitor of this enzyme. Chondroitinase B (dermatanase) cleaves the N-acetyl galatatosaminide bond bound to L-iduronic acid of dermatan sulfate in an elimination reaction, and has a Δ 4-hexuronic acid residue at the non-reducing end. Produces oligosaccharides (disaccharides and tetrasaccharides). This enzyme does not contain L-iduronic acid! /, Does not act on chondroitin sulfate A and chondroitin sulfate C. Dermatan, a derivative of dermatan sulfate with the sulfate group removed, is not a substrate for this enzyme. The site where the second position of the L-iduronic acid unit of dermatan sulfate is sulfated is more cleaved by this enzyme. Chondroitinase ABC cleaves the N-acetyl hexosaminide bond of chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, chondroitin and hyaluronic acid in an elimination reaction, and Δ 4- Mainly produces disaccharides with hexuronic acid residues. This enzyme does not act on ketalan sulfate, heparin and heparan sulfate. Chondroitinase is a collective term for these enzymes that have different properties but share the same properties of mucopolysaccharide-degrading enzymes, and are not limited to one row and seven rows of Cnondroitinase ACI, then hondroitinase A and II, and then hondrotinase. Not limited to B, Chond roitinase ABC.

ί列 は、 chondroitin— 4— sulfatase、 chondroitin— 6— sulfataseは、と もにゲノムデータベース上の複数のァクセッション番号で参照される配列（例えば Gen bankァクセッション番号 NT_039500 (その一部はァクセッション番号 CAAA01098429 ( 配列番号： 91)としてあらわされる）、 NT_078575、 NT_039353、 NW_001030904、 NW_0 01030811、 NW_001030796、 NW_000349)として公共遺伝子データベース Genbank上 で検索される。 [0048] Enzymes that share such characteristics do not necessarily correspond to one gene on genomic DNA.

The ί column is chondroitin—4—sulfatase, chondroitin—6—sulfatase, and sequences referenced by multiple accession numbers in the genome database (eg Gen bank accession number NT_039500 (some of which are (Accession number CAAA01098429 (SEQ ID NO: 91)), NT_078575, NT_039353, NW_001030904, NW_0 01030811, NW_001030796, NW_000349) are searched on the public gene database Genbank.

 [0049] Among those exemplified above, those corresponding to individual genes are shown as follows. That is, examples of the above chondroitin sulfate proteodaricans include aggrican, versican, neurocan, brevican ゝ β glycan, Decorm, Biglycan, Fibromodulin, P — Lb, an enzyme that cleaves or degrades chondroitin sulfate proteodarican, and these. ADAMTS-1, ADAMTS-4, ADAMTS-5, Calpain I, examples of enzymes involved in chondroitin synthesis, GalNAc4ST-1, GalNAc4ST-2, GALNAC4 S-6ST, UA20ST, GalT-1 C4ST-1, C4ST-2, C4ST-3, D4ST, C6ST-1, and C6ST-2, public genes of genes encoding in humans, exemplified as GalT-II, GlcAT-1, XylosylT, and sulfotransferase The accession number, base sequence and amino acid sequence in the database Genbank are as follows.

 aggrican (Accession number NM—007424, SEQ ID NO: 1 for nucleotide sequence, SEQ ID NO: 2 for amino acid sequence)

 versican (Accession number BC096495, SEQ ID NO: 3 for nucleotide sequence, SEQ ID NO: 4 for amino acid sequence)

neurocan (accession number NM—010875, nucleotide sequence SEQ ID NO: 5, amino acid sequence SEQ ID NO: 6) brevican (Accession number NM_007529, nucleotide sequence number: 7, amino acid sequence number: 8)

jS glycan (Accession number AF039601, nucleotide sequence number: 9, amino acid sequence number: 10)

 Decorin (accession number NM—007833, nucleotide sequence SEQ ID NO: 11, amino acid sequence SEQ ID NO: 12)

 Biglycan (Accession number BC057185, SEQ ID NO: 13 for nucleotide sequence, SEQ ID NO: 14 for amino acid sequence)

 Fibromodulin (Accession number NM—021355, nucleotide sequence number: 15, amino acid sequence number: 16)

 PG-Lb (Accession number NM—007884, nucleotide sequence number: 17; amino acid sequence number: 18)

 ADAMTS-1 (Accession number NM_009621, nucleotide sequence SEQ ID NO: 19, amino acid sequence SEQ ID NO: 20)

 ADAMTS-4 (Accession number NM—172845, SEQ ID NO: 21 of nucleotide sequence, SEQ ID NO: 22 of amino acid sequence)

 ADAMTS-5 (Accession number AF140673, nucleotide sequence SEQ ID NO: 23, amino acid sequence SEQ ID NO: 24)

 Calpain I (Accession number NM—007600, nucleotide sequence number: 25, amino acid sequence number: 26)

 GalNAc4ST-l (accession number NM—175140, nucleotide sequence SEQ ID NO: 27, amino acid sequence SEQ ID NO: 28)

 GalNAc4ST-2 (Accession number NM—199055, nucleotide sequence number: 29, amino acid sequence number: 30)

 GALNAC4S-6ST (Accession number NM_029935, nucleotide sequence SEQ ID NO: 31, amino acid sequence SEQ ID NO: 32)

UA20ST (Accession number NM—177387, SEQ ID NO: 33 for nucleotide sequence, SEQ ID NO: 34 for amino acid sequence) GalT-I (Accession number NM_016769, nucleotide sequence number: 35, amino acid sequence number: 36)

 GalT-11 (accession number BC064767, nucleotide sequence number: 37, amino acid sequence number: 38)

 GlcAT-I (Accession No. BC058082, nucleotide sequence SEQ ID NO: 39, amino acid sequence SEQ ID NO: 40, or accession number NM_024256, nucleotide sequence SEQ ID NO: 41, amino acid sequence SEQ ID NO: 42 )

 XylosylT (Accession number NM—145828, nucleotide sequence number: 43, amino acid sequence number: 44)

 C4ST-1 (Accession number NM— 021439, nucleotide sequence number: 45, amino acid sequence number: 46)

 C4ST-2 (Accession number NM—021528, nucleotide sequence SEQ ID NO: 47, amino acid sequence SEQ ID NO: 48)

 C4ST-3 (Accession No. XM—355798, nucleotide sequence SEQ ID NO: 49, amino acid sequence SEQ ID NO: 50)

 D4ST (accession number NM_028117, nucleotide sequence SEQ ID NO: 51, amino acid sequence SEQ ID NO: 52)

 C6ST-1 (Accession number NM—016803, SEQ ID NO: 53 of the nucleotide sequence, SEQ ID NO: 54 of the amino acid sequence)

 C6ST-2 (Accession number AB046929, nucleotide sequence number: 55, amino acid sequence number: 56)

Even proteins other than those described above have high homology (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more) with the sequences described in the sequence listing. In addition, a protein having the function of the protein (for example, a function of binding to a structural component in a cell) is included in the protein of the present invention. The above-mentioned protein is, if f row, IJ number: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 Amino acid sequence with one or more amino acids added, deleted, substituted or inserted A protein consisting of a sequence, wherein the number of normally changing amino acids is within 30 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, most preferably within 3 amino acids.

[0051] Examples of the gene in the present invention include, for example, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55. Etc.).

 [0052] SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,

 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55! In general, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 , 41, 43, 45, 47, 49, 51, 53, 55! /, And DNA with high displacement! High homology means 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more (e.g., 95% or more, further 96%, 97%, 98% or 99% or more). It means the homology of (above). This homology was determined by the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Aca d. Sci. USA 90: 5873- 7) can be determined by. In addition, when the DNA is isolated from the living body, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55 It is considered to be noblyzed under stringent conditions with DNA. Here, “stringent conditions” include, for example, “2 X SSC, 0.1% SDS, 50.C”, “2 X SSC, 0.1% SDS, 42 ° Cj,“ 1 X SSC, 0.1% SDS, 37 `` ° C '', more stringent conditions as `` 2 X SSC, 0.1% SDS, 65 ° C '', `` 0.5 X SSC, 0.1% SDS, 42 ° C '' and `` 0.2 X SSC, 0.1% SDS, 65 ° C '' Can be mentioned.

[0053] A person skilled in the art can convert a protein functionally equivalent to the above protein from the above highly homologous protein into an action of promoting the degradation of chondroitin sulfate proteodarican, a synthetic inhibitory action, a desulfation action, or It can be suitably obtained by using a method for measuring the activity of sulfate inhibitory action. A specific activity measuring method is the screen in the present invention described later. It is described in the section of the Jung method. Moreover, those skilled in the art can appropriately obtain an endogenous gene corresponding to the above gene in another organism based on the base sequence of the above gene. In the present specification, the above-mentioned proteins and genes corresponding to the above-mentioned proteins and genes in organisms other than humans, or the above-mentioned proteins and genes functionally equivalent to the above-mentioned proteins and genes are also simply referred to as It may be described in.

 [0054] The protein of the present invention can be prepared not only as a natural protein but also as a recombinant protein using a gene recombination technique. As a natural protein, for example, it can be prepared by a method using affinity chromatography using an antibody against the above protein against a cell (tissue) extract that is thought to express the above protein. It is. On the other hand, a recombinant protein can be prepared, for example, by culturing cells transformed with DNA encoding the protein. The above-mentioned protein of the present invention is suitably used, for example, in the screening method described later.

 [0055] "Nucleic acid" in the present invention means RNA or DNA. Chemically synthesized nucleic acid analogs such as so-called PNA (peptide nucleic acid) are also included in the nucleic acid of the present invention. PNA replaces the pentose / phosphate skeleton, which is the basic skeleton structure of nucleic acid, with a polyamide skeleton with glycine as a unit, and has a three-dimensional structure very similar to nucleic acid.

[0056] As a method for inhibiting the expression of a specific endogenous gene, a method using an antisense technique is well known to those skilled in the art. There are a number of factors that cause the antisense nucleic acid to inhibit the expression of the target gene. Inhibition of transcription initiation due to triplex formation, transcription inhibition due to hybridization with a site where an open loop structure was locally created by RNA polymerase, transcription inhibition due to hybridization with RNA undergoing synthesis, intron Inhibition of splicing by hybridization at the junction of Etason and etason, inhibition of splicing by spliceosome formation site, inhibition of splicing by hybrid formation, inhibition of nuclear force by hybridization with mRNA, inhibition of migration to cytoplasm, capping site and poly (A) Splicing inhibition by hybridization with additional site, translation initiation inhibition by hybridization with translation initiation factor binding site, ribosome binding near the initiation codon Inhibition of translation by hybridization with a site, inhibition of elongation of a peptide chain by hybridization with a translation region of mRNA or a polysome binding site, and inhibition of gene expression by hybridization with an interaction site between a nucleic acid and a protein. In this way, antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shinsei Kagaku Kenkyusho 2 Nucleic acid IV gene replication and expression, Japan Biochemical Society, Tokyo Chemical Doujin, 1993, 319-347.).

The antisense nucleic acid used in the present invention encodes any one of the above-described chondroitin sulfate proteodarican core protein, synthase, desulfase-inhibiting protein, and sulfotransferase by any of the above-described actions. Gene expression and Z or function may be inhibited. In one embodiment, the above-mentioned chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, or a gene encoding a sulfate transfer enzyme is complementary to the untranslated region near the 5 'end of the mRNA. If an antisense sequence is designed, it would be effective to inhibit gene translation. In addition, a sequence complementary to the coding region or the 3 ′ untranslated region can also be used. As described above, the anti-translation region of the anti-translation region consisting of the core protein, the synthase, the desulfation enzyme inhibitory protein, or the gene encoding the sulfotransferase as described above is not limited to the anti-translation region. A nucleic acid containing a sense sequence is also included in the antisense nucleic acid used in the present invention. The antisense nucleic acid to be used is linked downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The nucleic acid thus prepared can be transformed into a desired animal (cell) by using a known method. The sequence of the antisense nucleic acid is the gene encoding the endogenous chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, or sulfotransferase of the animal (cell) to be transformed or one of them. It is preferable that the sequence is complementary to the region, but it may not be completely complementary as long as the expression of the gene can be effectively suppressed. The transcribed RNA has a complementarity of preferably 90% or more, and most preferably 95% or more, to the target gene transcript. In order to effectively inhibit the expression of a target gene using an antisense nucleic acid, the length of the antisense nucleic acid is preferably at least 15 bases and less than 25 bases, but the antisense nucleic acid of the present invention is indispensable. The length is not limited to this length, and may be, for example, 100 bases or more, or 500 bases or more.

 [0058] The antisense nucleic acid of the present invention is not particularly limited. For example, the base sequence of Versican gene (GenBank accession number BC096495, SEQ ID NO: 3), the base sequence of C4ST-1 gene (GenBank accession) No. NM—021439, SEQ ID NO: 45), C4ST-2 gene base sequence (GenBank accession number NM—0221528, SEQ ID NO: 47), C4ST-3 gene base sequence (GenBank accession) No. XM-355798, SEQ ID NO: 49).

 [0059] Inhibition of the expression of the above chondroitin sulfate proteodarican core protein, synthase, desulfurization enzyme inhibitory protein, or gene encoding sulfotransferase uses ribozyme or DNA encoding ribozyme It is also possible to do this. Ribozyme refers to an RNA molecule that has catalytic activity. Among the abilities of ribozymes having various activities, research focusing on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes that cleave RNA site-specifically. Some ribozymes have a size of 400 nucleotides or more, such as the group I intron type and Ml RNA contained in RNase P, and some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 19 90, 35, 2191.).

[0060] For example, the self-cleaving domain of the hammerhead ribozyme has the ability to cleave 3 'of C15 in the sequence G13U14C15. For its activity, base pairing between U14 and A9 is important. It has been shown that A15 or U15 can also be cleaved (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.) ₀ Designing a ribozyme whose substrate binding site is complementary to the RNA sequence near the target site Thus, a restriction enzyme-like RNA cleavage ribozyme that recognizes the sequence UC, UU, or UA in the target RNA can be generated (Koizumi, M. et al., FEBS Lett, 1988, 239, 285. Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191., Koizumi, M. et al, Nucl Acids Res, 1989, 17, 7059.).

[0061] Hairpin ribozymes are also useful for the purposes of the present invention. This ribozyme is found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, JM., Nature, 1986, 323, 349.). It has been shown that target-specific RNA cleavage ribozymes can also be generated from hairpin ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.). In this way, the ribozyme is used to specifically cleave the above-mentioned chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, or transcript of a gene encoding a sulfotransferase. Gene expression can be inhibited.

 [0062] Inhibition of endogenous gene expression can also be achieved by RNA interference (hereinafter abbreviated as "RNAi") using double-stranded RNA having the same or similar sequence as the target gene sequence. It can be carried out.

 [0063] With the completion of recent genome projects, human base sequences have been deciphered and many disease-related genes have been actively identified. Currently, therapeutic methods and drug discovery targeting specific genes are being actively implemented. ing. Of particular interest is the application of small interfering RNA (siRNA), which exerts a specific post-transcriptional inhibitory effect, to gene therapy. RNAi is a technique that is currently attracting attention because when a double-stranded RNA (dsRNA) is taken directly into a cell, expression of a gene having a sequence homologous to this dsRNA is suppressed. In mammalian cells, RNAi can be induced by using short dsRNA (siRNA). RNAi is more stable, easier to experiment, and less expensive than knockout mice. It has many advantages.

[0064] A nucleic acid having an inhibitory action by the RNAi effect is generally also referred to as siRNA or shRNA. RNAi is a sense RNA that has a sequence power that is homologous to the mRNA of the target gene, a complementary double-stranded antisense RNA, and a short double-stranded RNA that is powerful (hereinafter abbreviated as “dsRNA”). This is a phenomenon that induces destruction by specifically and selectively binding to the target gene mRNA, and efficiently inhibiting (suppressing) the expression of the target gene by cleaving the target gene. For example, when dsRNA is introduced into a cell, the expression of the gene homologous to the RNA is suppressed (knocked down). Since RNAi can suppress the expression of target genes in this way, it can be applied as a simple gene knockout method instead of the conventional complicated and low-efficiency gene disruption method by homologous recombination, or for gene therapy. It is attracting attention as a method. The RNA used for RNAi is the above chondroit It does not have to be completely identical to the gene encoding the core protein, synthetic enzyme, desulfurase inhibitor protein, or sulfotransferase, or a partial region of this gene. It is preferable to have.

 [0065] In designing siRNA, the target is not particularly limited as long as it is a gene encoding the above-mentioned chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, or sulfate transferase. It is possible to make any arbitrary region as a target candidate. For example, the base sequence of Versican gene (SEQ ID NO: 3), the base sequence of C4ST-1 gene (SEQ ID NO: 45), the base sequence of C4ST-2 gene (SEQ ID NO: 47), the base sequence of C4ST-3 gene ( It can be created based on SEQ ID NO: 49). More specifically, a partial region of the sequence can be a target candidate. For example, a partial region of the Versican gene base sequence (SEQ ID NO: 57), the base of the C4ST-1 gene Partial region of the sequence (SEQ ID NO: 58), partial region of the base sequence of the C4ST-2 gene (SEQ ID NO: 59), partial region of the base sequence of the C4ST-3 gene (SEQ ID NO: 60), C6ST -Partial region of the base sequence of 1 gene (SEQ ID NO: 61), partial region of the base sequence of C6ST-2 gene (SEQ ID NO: 62), partial region of the base sequence of GalNAc4ST-l gene (SEQ ID NO: 6 3), a partial region of the base sequence of the GalNAc4ST-2 gene (SEQ ID NO: 64), a partial region of the base sequence of GALNAC4S-6ST (SEQ ID NO: 65), and the like. More specifically, siRNA targeting the DNA sequences specifically shown by this specification (SEQ ID NOs: 71 to 73 and 80-90) can be exemplified.

 [0066] In order to introduce siRNA into a cell, a method in which siRNA synthesized in vitro is linked to plasmid DNA and introduced into the cell, a method of annealing two RNAs, or the like can be employed.

 [0067] The two RNA molecules may be molecules having a structure in which one end is closed, for example, siRNA (shRNA) having a hairpin structure. shRNA is called short hairpin RNA, and is an RNA molecule having a stem-loop structure so that a part of a single strand forms a complementary strand with another region. That is, molecules capable of forming a double-stranded RNA structure in the molecule are also included in the siRNA of the present invention.

[0068] Further, preferred embodiments of the present invention include Versican, C4ST-1, C4ST-2, C4ST-3 and the like. In an siRNA targeting the DNA sequence (SEQ ID NOs: 71 to 73 and 80 to 90) specifically shown by the present specification, which can be suppressed by the RNAi effect, for example, 1 Alternatively, even a double-stranded RNA with a structure in which a small number of RNAs are added or deleted encodes the above-mentioned chondroitin sulfate proteodarican core protein, synthase, desulfurase oxidase inhibitor protein, or sulfate transferase Any siRNA of the present invention may be used as long as it has a function of suppressing gene expression.

 [0069] The RNA used for RNAi (siRNA) does not have to be the same as the gene encoding the protein or a partial region of the gene. ) Preferred to have sex.

 [0070] The force of DICER (a member of the RNase III nucleolytic enzyme family) contacts double-stranded RNA, and the double-stranded RNA is small iterfering RNA or It is thought to be broken down into small fragments called siRNA! / The double-stranded RNA having the RNAi effect in the present invention includes double-stranded RNA before being digested by DICER as described above. That is, even a long-chain RNA that does not have an RNAi effect with the same length is expected to be decomposed into siRNA having an RNAi effect by the cell. The length of the double stranded RNA is not particularly limited.

 [0071] For example, the above-mentioned chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, or two long-chains corresponding to the full-length region or almost the full-length region of the mRNA encoding the gene encoding sulfotransferase For example, the strand RNA can be decomposed in advance with DICER, and the degradation product can be used as the agent of the present invention. This degradation product is expected to contain double-stranded RNA molecules (siRNA) having the RNAi effect. According to this method, it is not necessary to particularly select a region on mRNA expected to have an RNAi effect. That is, the region on the mRNA of the above-mentioned gene of the present invention having an RNAi effect does not necessarily need to be accurately defined.

[0072] The above-mentioned "double-stranded RNA that can be suppressed by the RNAi effect" of the present invention means that, for those skilled in the art, the above-mentioned chondroitin sulfate proteoglycan core protein, synthase, and desulfase that are targets of the double-stranded RNA It can be appropriately prepared based on the base sequence of the gene encoding the inhibitory protein or sulfate transferase. For example, SEQ ID NO: 71 The double-stranded RNA of the present invention can be prepared based on the base sequence described in 1. That is, based on the nucleotide sequence set forth in SEQ ID NO: 71, an arbitrary continuous RNA region of mRNA that is a transcription product of the sequence is selected, and a double-stranded RNA corresponding to this region is prepared. The person skilled in the art can appropriately carry out within the range of normal trials. Moreover, those skilled in the art can also appropriately select a siRNA sequence having a stronger RNAi effect from the mRNA sequence that is a transcription product of the sequence, by a known method. Further, if one strand is known, those skilled in the art can easily know the base sequence of the other strand (complement). A siRNA can be appropriately prepared by those skilled in the art using a commercially available nucleic acid synthesizer. In addition, for synthesis of desired RNA, a general synthetic contract service can be used.

 [0073] The siRNA in the present invention may be a mixture of a plurality of sets of double-stranded RNAs for a region containing a target sequence, which need not necessarily be a set of double-stranded RNAs for the target sequence. Here, siRNA as a nucleic acid mixture corresponding to the target sequence can be appropriately prepared by a person skilled in the art using a commercially available nucleic acid synthesizer and a DICER enzyme. You can use the composite contract service. The siRNA of the present invention includes so-called “cocktail siRNA”.

 [0074] In addition, in the siRNA of the present invention, not all nucleotides are necessarily ribonucleotides (RNA). That is, in the present invention, one or more ribonucleotides constituting siRNA may be a corresponding deoxyribonucleotide. This “corresponding” refers to the same base species (adenine, guanine, cytosine, thymine (uracil)) although the structures of the sugar moieties are different. For example, a deoxyribonucleotide corresponding to a ribonucleotide having adenine refers to a deoxyribonucleotide having adenine. The “plurality” is not particularly limited, but preferably refers to a small number of about 2 to 5

[0075] Furthermore, a DNA (vector) capable of expressing the RNA of the present invention is also included in a preferable embodiment of the compound capable of suppressing the expression of the gene encoding the protein of the present invention. For example, the DNA (vector) capable of expressing the double-stranded RNA of the present invention is a DNA encoding one strand of the double-stranded RNA and a DNA encoding the other strand of the double-stranded RNA, Each DNA has a structure linked to a promoter so that it can be expressed. Those skilled in the art can appropriately prepare the DNA of the present invention by a general genetic engineering technique. More specifically, the expression vector of the present invention can be prepared by appropriately inserting DNA encoding the RNA of the present invention into various known expression vectors.

 [0076] In addition, the expression inhibitory substance of the present invention includes the above-mentioned chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, or expression regulatory region of a gene encoding a sulfotransferase (for example, Specific examples include the base sequence represented by SEQ ID NO: 66, which is the promoter region of PG-Lb.) By binding to the above-mentioned core protein of chondroitin sulfate proteodarican A compound that inhibits the expression of a gene encoding a synthase, a desulfase inhibitor protein, or a sulfotransferase. The compound is, for example, a promoter DNA fragment of a gene encoding the above chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, or sulfotransferase, and binding activity to the DNA fragment It can be obtained by a screening method using as an index. In addition, those skilled in the art will determine whether or not the desired compound inhibits the expression of the above-mentioned chondroitin sulfate-teododalican core protein, synthase, desulfase-inhibiting protein, or gene encoding sulfotransferase. The determination can be appropriately carried out by a known method such as a reporter assay method.

[0077] Further, the DNA (vector) capable of expressing the RNA of the present invention is also the above-described core protein, synthetic enzyme, desulfase-inhibiting protein, or sulfate group of the chondroitin sulfate proteodalycan of the present invention. Preferred embodiments of compounds capable of inhibiting the expression of genes encoding transferases are included in the embodiments. For example, the DNA (beta) capable of expressing the double-stranded RNA of the present invention is a DNA that encodes one strand of the double-stranded RNA and a DNA force that encodes the other strand of the double-stranded RNA, respectively. It is a DNA having a structure linked to a promoter so that it can be expressed. The above-mentioned DNA of the present invention can be appropriately prepared by those skilled in the art using a general genetic technique. More specifically, the expression vector of the present invention is inserted by appropriately inserting DNA encoding the RNA of the present invention into various known expression vectors. It is possible to produce a kuta.

 [0078] A preferred embodiment of the vector of the present invention is a vector that expresses RNA (siRNA) capable of suppressing the expression of Versican, C4ST-1, C4ST-2, C4ST-3, and the like by the RNAi effect. it can.

 [0079] The above-mentioned chondroitin sulfate proteodarican core protein, synthase, desulfation enzyme inhibitor compound, or antibody that binds to a sulfotransferase can be prepared by methods known to those skilled in the art. . A polyclonal antibody can be obtained, for example, as follows. Serum is obtained by immunizing small animals such as rabbits with recombinant (recombinant) protein expressed in microorganisms as a fusion protein with the above-mentioned natural protein or GST, or a partial peptide thereof. For example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, core protein of the above chondroitin sulfate proteodarican, synthase, desulfase inhibitor compound, or sulfate transferase Or by purification using a utility column coupled with a synthetic peptide. In the case of a monoclonal antibody, for example, the above-mentioned chondroitin sulfate proteodarican core protein, synthetic enzyme, desulfase inhibitor compound, or sulfotransferase or its partial peptide is immunized to a small animal such as a mouse. The spleen is removed from the mouse, ground and separated to separate the cells, and the cells and mouse myeloma cells are fused using a reagent such as polyethylene glycol, and the resulting fused cells (hybridoma) A clone that produces an antibody that binds to the above chondroitin sulfate proteoglycan coprotein, synthase, desulfase inhibitor compound, or sulfotransferase is selected. Next, the obtained noci / hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, It can be prepared by purification using the above-mentioned chondroitin sulfate proteodarican core protein, synthetic enzyme, desulfating enzyme inhibitory compound, or a protein column coupled with a sulfotransferase protein or a synthetic peptide. Is possible.

[0080] The antibody of the present invention binds to the above-described chondroitin sulfate proteodarican core protein of the present invention, a synthase, a desulfase inhibitor compound, or a sulfotransferase. As long as there is no particular limitation, in addition to the above polyclonal antibody and monoclonal antibody, a human antibody, a humanized antibody by genetic recombination, an antibody fragment thereof, or an antibody modification product thereof may be used.

 [0081] The protein of the present invention used as a sensitizing antigen for obtaining an antibody is not limited with respect to the animal species from which it is derived, but a protein derived from a mammal such as a mouse is preferred, and a protein derived from a human is particularly preferred. . A human-derived protein can be appropriately obtained by those skilled in the art using the gene sequence or amino acid sequence disclosed in the present specification.

 [0082] In the present invention, the protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein. Examples of the partial peptide of the protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein. As used herein, “antibody” means an antibody that reacts with the full length or fragment of a protein.

 [0083] In addition to immunizing non-human animals with antigens to obtain the above hyperidoma, human lymphocytes such as human lymphocytes infected with EB virus are sensitized in vitro with proteins, protein-expressing cells or lysates thereof. And fusion of sensitized lymphocytes with human-derived permanent mitotic cells, such as U266, to produce a hyperidoma that produces the desired human antibody with protein-binding activity. .

 [0084] The above-mentioned chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor compound, or antibody to sulfate group transferase of the present invention binds to the protein to thereby regulate the expression or function of the protein. An inhibiting effect is expected. When the obtained antibody is used for the purpose of administering it to the human body (antibody therapy), a human antibody or a humanized antibody is preferred in order to reduce immunogenicity.

[0085] Further, the present invention relates to the above chondroitin sulfate proteodarican as a substance capable of inhibiting the function of the core protein, synthase, desulfase inhibitor, or sulfotransferase of the above chondroitin sulfate proteodarican. It also contains a low molecular weight substance (low molecular weight compound) that binds to a core protein, a synthetic enzyme, a desulfurizing oxidase inhibiting compound, or a sulfotransferase. The low molecular weight substance may be a natural or artificial compound. Through Usually, it is a compound that can be produced or obtained by using methods known to those skilled in the art. The compound of the present invention can also be obtained by the screening method described later.

 [0086] Furthermore, as a substance capable of inhibiting the expression or function of the above-mentioned chondroitin sulfate proteodarican core protein, synthetic enzyme, desulfase inhibitor protein, or sulfotransferase of the present invention, the above-mentioned chondroitin sulfate proteodarican And a mutant having a dominant negative property (dominant negative protein) with respect to a core protein, a synthase, a desulfase inhibitor protein, or a sulfotransferase. “The chondroitin sulfate proteodarican core protein, synthase, desulfurase inhibitor protein, or the protein variant having a dominant negative property to sulfate group” refers to the core of chondroitin sulfate proteodarican. It refers to a protein having a function of eliminating or reducing the activity of an endogenous wild-type protein by expressing a gene encoding a protein, a synthase, a desulfase-inhibiting protein, or a sulfotransferase. Examples of such dominant negative proteins include Versican core protein mutants that competitively inhibit the binding to chondroitin sulfate with the wild-type Versican core protein.

 [0087] In the present invention, the organ, tissue, or cell that inhibits the production or accumulation of chondroitin sulfate proteodarican is not particularly limited, but is preferably an organ, tissue, or cell that secretes insulin, or an organ that acts on insulin. A tissue or cell, more preferably a spleen β-cell.

 [0088] A compound that inhibits the production or accumulation of chondroitin sulfate proteodarican is expected to be a drug for the treatment or prevention of insulin resistance diseases. Here, “treatment or prevention” refers to a case where it is not necessary to have a complete therapeutic or preventive effect on an organ, tissue, or cell that exhibits insulin resistance. It's okay.

[0089] In the present invention, the insulin resistance disease is not particularly limited as long as it is a disease associated with insulin resistance, but preferably a disease exhibiting insulin resistance associated with metabolic syndrome (visceral fat syndrome), Non-insulin dependent (type 2) diabetes and the like. Ma Further, metabolic syndrome such as obesity, hyperlipidemia, arteriosclerosis, and complications such as fatty liver disorder and visceral fat are also included in the insulin resistance disease of the present invention.

 [0090] The insulin resistance inhibitor of the present invention has an action of suppressing insulin resistance by inhibiting the production or accumulation of chondroitin sulfate proteodalycan, which is the cause of insulin resistance. Therefore, as a preferred embodiment of the present invention, for example, a metabolic syndrome therapeutic agent or a non-insulin dependent (type 2) diabetes therapeutic agent comprising the insulin resistance inhibitor of the present invention as an active ingredient is provided.

 In addition, the “insulin resistance inhibitor” of the present invention can also be expressed as “insulin resistance therapeutic agent”, “insulin resistance improving agent” or the like. In the present invention, the “inhibitor” can also be expressed as “medicine”, “pharmaceutical composition”, “therapeutic drug”, and the like.

 It should be noted that the “treatment” in the present invention includes a preventive effect that can suppress the occurrence of insulin resistance in advance. In addition, it is not necessarily limited to the case where the insulin resistance-expressing cell (tissue) has a complete therapeutic effect, and may be a case where it has a partial effect.

 [0093] The drug of the present invention can be mixed with a physiologically acceptable carrier, excipient, diluent or the like, and can be administered orally or parenterally as a pharmaceutical composition. As oral preparations, dosage forms such as granules, powders, tablets, capsules, solvents, emulsions or suspensions can be used. As a parenteral preparation, a dosage form such as an injection, a drip infusion, an external medicine, or a suppository can be selected. Examples of injections include subcutaneous injections, intramuscular injections, and intraperitoneal injections. The topical drug may be a nasal agent or an ointment. The preparation technique of the above dosage form so as to include the drug of the present invention as the main component is known.

[0094] For example, a tablet for oral administration can be produced by adding an excipient, a disintegrant, a binder, a lubricant and the like to the drug of the present invention, and mixing and compression-molding. As the excipient, lactose, starch, mannitol or the like is generally used. As the disintegrant, calcium carbonate or carboxymethyl cellulose calcium is generally used. As the binder, gum arabic, carboxymethylcellulose, or polyvinylpyrrolidone is used. As the lubricant, talc magnesium stearate and the like are known. [0095] The tablet containing the drug of the present invention can be subjected to a known coating for masking or enteric preparation. As the coating agent, ethyl cellulose, polyoxyethylene glycol or the like can be used.

 [0096] In addition, an injection can be obtained by dissolving the agent of the present invention as a main component together with an appropriate dispersant, or dissolving or dispersing in a dispersion medium. Depending on the choice of the dispersion medium, either aqueous solvent or oil-based solvent can be used. In order to use an aqueous solvent, distilled water, physiological saline, Ringer's solution, or the like is used as a dispersion medium. For oil-based solvents, various vegetable oils such as propylene glycol are used as dispersion media. At this time, a preservative such as paraben can be added as necessary. In the injection, a known isotonic agent such as sodium chloride or glucose can be added. In addition, a soothing agent such as salt benzalcoum can be added.

 [0097] Further, an external preparation can be obtained by making the agent of the present invention into a solid, liquid, or semi-solid composition. About a solid or liquid composition, it can be set as an external preparation by setting it as the composition similar to what was described previously. A semi-solid composition can be prepared by adding a thickener to an appropriate solvent as required. As the solvent, water, ethyl alcohol, polyethylene glycol, or the like can be used. As the thickener, bentonite, polybutyl alcohol, acrylic acid, methacrylic acid, polyvinylpyrrolidone, or the like is generally used. To this composition can be added a preservative such as salt benzalkonium. A suppository can also be obtained by combining an oily base material such as cacao butter or an aqueous gel base material such as cellulose derivative as a carrier.

 [0098] In the case of using the drug of the present invention as a gene therapy agent, in addition to the method of directly administering the drug of the present invention by injection, a method of administering a vector incorporating a nucleic acid can be mentioned. Examples of the above-mentioned vectors include adenovirus vectors, adeno-associated virus vectors, herpes vinores vectors, vaccinia winores betaters, retro winores betaters, and lentivirus vectors. Can be invested well.

[0099] It is also possible to introduce the drug of the present invention into a phospholipid vesicle such as a ribosome and administer the vesicle. Endoplasmic reticulum with siRNA or shRNA retained in the lipofusion method Introduce into a predetermined cell. The obtained cells are then administered systemically, for example, intravenously or intraarterially. It can also be administered locally to an insulin resistant tissue or the like. siRNA has a very excellent specific post-transcriptional repressive effect in vitro.In vivo, it is rapidly degraded by the nuclease activity in serum and is therefore more optimal and effective due to its limited duration. Development of a new delivery system has been demanded. As an example, the biocompatible material atelocollagen is mixed with nucleic acid from Ochiya, T et al. Nature Med., 5: 707-710, 1999, Curr. Gene Ther., 1: 31-52, 2001. However, when a complex is formed, it has been reported that the ability of degrading enzymes in the living body also protects nucleic acids and is a very suitable carrier for siRNA. This is not a limitation.

[0100] The necessary amount (effective amount) of the drug of the present invention is administered to mammals including humans within the range of safe doses. The dosage of the drug of the present invention can be appropriately determined finally based on the judgment of a doctor or veterinarian in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like. For example, the power varies depending on age, sex, symptoms, administration route, number of administrations, and dosage forms.For example, the dose in the case of adenovirus is about 10 ⁶ to 10 ¹³ per day, 1 week to 8 It is administered at weekly intervals.

 [0101] Further, in order to introduce siRNA or shRNA into a target tissue or organ, a commercially available gene introduction kit (for example, Adeno Express: Clontech) can also be used.

 [0102] When the drug of the present invention is used, the application site or the type of the disease is not particularly limited as long as it is a disease that expresses insulin resistance. For example, diabetes, obesity, hyperlipidemia, arteriosclerosis, hypertension, etc. Applies to The above diseases may be concurrent with other diseases.

 [0103] The present invention also provides a method for screening an insulin resistance inhibitor, which comprises selecting a substance having an action of inhibiting the production or accumulation of test sample chondroitin sulfate proteodarican. By the screening method of the present invention, a candidate compound for an insulin resistance inhibitor or an insulin resistance inhibitor can be efficiently obtained.

[0104] A preferred embodiment of the screening method of the present invention is any of the following (a) to (d): A method for screening an insulin resistance inhibitor, comprising a step of selecting a substance having the above action.

( _a ) Promoting the degradation of chondroitin sulfate proteodarican

 (b) Inhibition of chondroitin sulfate proteodarican synthesis

 (c) Desulfation effect of chondroitin sulfate proteodarican

 (d) Sulfate inhibitory action of chondroitin sulfate proteodarican

 [0105] As a basic principle of screening common to these, a typical example includes one including the following steps.

 (1) Chondroitin sulfate proteodarican (CSPG) itself Z or glycosaminoglycan (GAG) chain Z or cells that synthesize (generate) CSPG or GAG chain

 (2) Test compounds (for example, huge compound libraries owned by pharmaceutical companies)

 (3) Cut section of chondroitin sulfate proteodarican (CSPG) Z chondroitin sulfate proteodarican (CSPG) amount Z method for detecting the amount of free glycosaminodarlican (GAG) The above three tools are used. It is desirable to mix (1) and (2) in a test tube or on a culture dish, and detect the effect simply by (3).

 [0106] Hereinafter, embodiments of the screening method of the present invention will be exemplified. In the embodiment described below, the chondroitin sulfate proteodarican, synthase, desulfase inhibitor compound, sulfate transferase, degradation promoting enzyme, and desulfase used are derived from human, mouse, Forces derived from rats and the like are not particularly limited to those derived from these. The part of chondroitin sulfate proteodalycan is a component such as a glycosaminodarican chain, a core protein, or a part thereof, and is not particularly limited.

 [0107] The test compound used in the embodiments described below is not particularly limited. For example, a single compound such as a natural compound, an organic compound, an inorganic compound, a protein, and a peptide, a compound library, Examples include gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, and the like.

[0108] In addition, the "contact" to the test compound in the embodiment described below is usually chondroitin sulfate proteodarican, a part thereof, a synthase, a desulfase inhibitor compound, a sulfotransferase, a degradation promoting enzyme. Or by mixing desulfating enzyme with test compound iS Not limited to this method. For example, the above “contact” can be performed by contacting a cell expressing these proteins or a part thereof with a test compound.

[0109] The origin of the "cell" in the embodiments described below includes cells derived from humans, mice, rats, etc., but is not particularly limited to cells derived from these, and in each embodiment It is also possible to use microbial cells such as Escherichia coli and yeast transformed to express the protein to be used once. For example, a cell expressing a chondroitin sulfate proteodarican can be expressed as a cell that expresses an endogenous chondroitin sulfate proteodarican gene or an exogenous chondroitin sulfate proteodarican gene, Cells in which the gene is expressed can be used. A cell in which an exogenous chondroitin sulfate proteodarican gene is expressed can be usually prepared by introducing an expression vector into which a chondroitin sulfate proteodarican gene is inserted into a host cell. The expression vector can be produced by a general genetic engineering technique.

 [0110] In the following description, the term "chondroitin sulfate proteodarican core protein" means, for example, a matrix type chondroitin sulfate proteoglycan, a core protein such as aggrican, vers ican, neurocan, brevican, or a membrane type chondroitin sulfate. Proteoglycans are core proteins such as Decorin, Biglycan, Fibromodulin, and PG-Lb. Examples of the “synthetic enzyme” include GalNAc4ST-1, GalNAc4ST-2, GALNAC4S-6ST, UA20ST, GalT-1, GalT-II, GlcAT-1, and XylosylT. “Sulfyltransferase” includes, for example, C4ST-l (Chondroitin D—N—acetylgalactosamine—4—0—sulfotransfer ase 1), C4b Γ—2 (and honaroitin D—N—acetylgalactosamine—4—0—sulfotransferase 2). ), C4 ST-3 (Chondroitin DN-acetylgalactosamine-4-0-sulfotransferase 3), D4ST, C6ST-1, C6ST-2, and the like. Examples of the “degradation promoting enzyme” include ADAMTS-1, ADAMTS-4, ADAMTS-5, honaroitinase ABC (and hABC) ゝ Cnondroitinase AC, Chondroitinase B, Calpain I and the like. Examples of the “desulfating enzyme” include Chondroitin-4-sulfatase and nondroitm-sulfatase.

[0111] As an embodiment of the screening method of the present invention, there may be mentioned a method comprising a step of selecting a compound having an action of promoting the degradation of chondroitin sulfate proteodarican. Main departure For example, the above-described method has the following process power.

( _a ) The process of contacting the test compound with chondroitin sulfate proteodarican or a part thereof

 (b) a step of measuring the abundance of chondroitin sulfate proteodarican or a part thereof

(c) A step of selecting a substance that reduces the abundance compared to the case where measurement is performed in the absence of the test compound.

 [0112] In the above method, first, a test compound is brought into contact with chondroitin sulfate proteodarican or a part thereof.

 [0113] Next, in the present method, the amount of chondroitin sulfate proteodarican or a part thereof is measured. The measurement can be performed by methods known to those skilled in the art. For example, it can be detected by measuring the amount of labeling using a labeled compound or antibody that binds to chondroitin sulfate proteodarican or a part thereof. It can also be detected using a chromatographic method or mass spectrometry.

[0114] In this method, a compound that reduces the abundance of the chondroitin sulfate proteodarican or a part thereof is then selected as compared with the case where the test compound is not contacted (control). The compound that lowers becomes a drug for the treatment of insulin resistance.

[0115] A simple example of a method and a specific example that can be evaluated (measured) as to whether or not the test compound has the activity of the above-described (a) degradation promoting action is shown below.

[0116] (a) Screening method for promoting the degradation of chondroitin sulfate proteodarican

 CS-GAG includes chondroitin sulfate A (CS-A), CS-B, CS-C (Seikagaku Corporation, ICN, Sigma, etc.), human-derived proteodalycan (BGN, ISL, etc.), etc. Prepare and coat 96 well plates at a concentration of 10 μg / mL (Kawashima H et al .; J. Biol. Chem. 277: 12921-12930, 2002. etc.). Add various test compounds to each well of this plate and detect CS-GAG change after 2 hours reaction at 37 ° C.

[0117] As a detection method, for example, a WFA lectin (Nodafuji lectin) binding method can be mentioned as a simple method. Since WFA lectin binds to the GalNAc residue of CS-GAG chain, CS-GAG can be easily detected. As a positive control for the test compound, chondroitinase ( Chondroitinase) Use ABC. If the CS-GAG chain is degraded by chondroitinase ABC, the WFA lectin cannot be bound. More specifically, FITC-labeled WFA lectin (such as EY) is added to the CS coating well before and after the test compound is mixed, and the CS-GAG is decomposed, so that the FITC fluorescence in the tool Changes in intensity can be quantitatively and easily determined using a detection device such as a fluorescence plate reader or a fluorescence microscope. The compound with the lowest fluorescence value before and after mixing can be determined as a novel therapeutic candidate compound that satisfies this concept.

 [0118] As another detection method, an anti-CS antibody (clone: CS56, manufactured by Seikagaku Corporation) that directly labels CS-GAG itself can be used. As with the WFA lectin, FITC-labeled anti-CS antibody can be added to CS-coated wells so that mass screening can be performed in a very short time and simply if changes in fluorescence values are observed.

 [0119] As a more detailed detection method, use the sGA G Assay Kit (manufactured by WIESLAB), Sulphanated Glycosaminoglycans, ELISA Kit (manufactured by FUNAK OSHI), etc., using the plate before and after mixing the test compound as it is. There is a method to accurately quantify and quantify the GAG content.

 [0120] More specifically, free GAG can be obtained by adding 2-AB (2-aminobenzamide) or 2-AP (2-aminopyridine, both of which are manufactured by LUD) to the plate before and after mixing of the test compound. More detailed analysis is possible by simply fluorescently labeling the reducing end of the chain and analyzing each type of sugar chain and the content of each type by HPLC, MALDI-MS, LC-MS, etc. . This is a method for the next stage of screening in which the properties of candidate compounds are examined in detail.

 [0121] As another embodiment of the screening method of the present invention, a method including a step of selecting a substance having an inhibitory action on chondroitin sulfate proteodarican synthesis can be mentioned. The above-described method of the present invention also has the following process power, for example.

( _a ) A test compound is brought into contact with a cell group expressing chondroitin sulfate proteodarican or a part thereof, a cell extract, or a substance group containing an enzyme and a substrate constituting the synthesis process of chondroitin sulfate proteodarican. Process

(b) a step of measuring the amount of chondroitin sulfate proteoglycan or an intermediate in the synthesis process in the cell, cell extract or substance group. (c) The test compound is not brought into contact with the test compound!

 [0122] In the above method, first, a cell that expresses chondroitin sulfate proteodarican or a part thereof, a cell extract, or a group of substances including an enzyme and a substrate constituting the synthesis process of chondroitin sulfate proteodarican, and the like. A test compound is brought into contact.

 [0123] Next, the synthesis amount of chondroitin sulfate proteodarican or an intermediate in the synthesis process is measured. The measurement can be appropriately carried out by those skilled in the art by a known method, for example, a method using a labeled antibody, mass spectrometry, chromatography, or the like.

 [0124] Furthermore, a compound that reduces (suppresses) the synthesis amount is selected as compared with the case where the test compound is not contacted! Compounds that decrease (suppress) become drugs for the treatment of insulin resistance.

 [0125] A simple example of a method and a specific example that can be evaluated (measured) as to whether or not the test compound has the activity of the above-mentioned (b) synthesis inhibitory action is shown below.

[0126] (b) Screening for the synthetic inhibitory action of chondroitin sulfate proteodarican Method aspect:

 Cells and cell lines that synthesize chondroitin sulfate are known to the investigator. In humans, for example, chondroitin sulfate is produced in 16 hours of cell culture by the standard method of collecting and culturing mononuclear cells after collecting peripheral blood from healthy individuals (Uhlin-Hansen L et al. , Blood 82: 2880, 1993.). More simply, known cell lines such as fibroblast cell line NIH3T3 (Phillip HA, et al. J. Biol. Chem. 279: 48640, 2004), renal tubule-derived cancer cell line ACHN (Kawashima H et al., J. Biol. Chem. 277: 12921, 2002), distal renal tubule-derived cell line MDCK (Borges FT et al., Kidney Int. 68: 1630, 2005., etc.), vascular endothelial cells Many strains such as HUVEC strains (Schick BP et al., Blood 97: 449, 2001, etc.) are mentioned. Various test compounds are mixed during the process of culturing such a cell line for a certain period of time, and the change in the amount of CS-GAG before and after the culture can be easily numerically measured by the method (a). Compound power that suppresses the increase in CS-GAG amount after cell culture (ie, reflects the amount of CS-GAG synthesis) It can be easily determined as a therapeutic candidate compound that satisfies this concept.

[0127] Furthermore, more selectively, for example, CS-GAG synthases such as GalNAc4ST-l and XylosylT Can be made into CHO cells and L cells by a well-known method, and a cell line in which the gene is constitutively expressed can be prepared. By using such a cell line that constantly synthesizes CS-GAG, it is possible to more clearly determine the candidate treatment compound.

[0128] As another aspect of the screening method of the present invention, a method including a step of selecting a substance having a desulfating action of chondroitin sulfate proteodarican can be mentioned. The above-described method of the present invention also has the following process power, for example.

( _a ) The process of contacting the test compound with chondroitin sulfate proteodarican or a part thereof

 (b) a step of measuring the amount of chondroitin sulfate proteodarican or a portion of the sulfate which has been received.

 (c) A step of selecting a substance that receives sulfate and reduces the amount thereof compared to the case of measurement in the absence of the test compound.

 [0129] In the above method, first, a test compound is brought into contact with chondroitin sulfate proteodarican or a part thereof.

 [0130] Next, in the present method, the amount of chondroitin sulfate proteodarican or a part thereof that has received sulfate is measured. The measurement can be performed by methods known to those skilled in the art. For example, it can be detected by measuring the amount of labeling using a labeled compound or antibody that binds to the structure of desulfurization oxidation remaining in chondroitin sulfate proteodarican or a part thereof. It can also be detected using chromatography, mass spectrometry, and the like.

 [0131] In this method, a compound that reduces the abundance of the chondroitin sulfate proteodarican or a part thereof is then selected as compared with the case where the test compound is not contacted (control). The compound that lowers becomes a drug for the treatment of insulin resistance.

[0132] A simple example of a method and a specific example that can be evaluated (measured) depending on whether or not the test compound has the above-mentioned (c) activity of desulfation activity is shown below. .

[0133] (c) Screening for the desulfation effect of chondroitin sulfate proteodarican Method aspect:

Basically, as in the method (a) above, human-derived proteodaricans (BGN, ISL, etc.), etc. And coated on a 96-well plate at a concentration of 10 μg / mL (Kawashima H et al .; J. Biol. Chem. 277: 12921-12930, 2002., etc.). Add various test compounds to each well of this plate and detect CS-GAG change after 2 hours reaction at 37 ° C.

 [0134] The detection method was carried out by desulfating the disaccharide structure of the desulfated fragment remaining on the core protein side of the proteodarican into the anti-proteodarican A di4S antibody (clone; 2-B-6, 4 Recognize the part that received sulfate at the position) or anti-proteodarican Δdi6S (clone; 3-B-3, recognize the part that received sulfate at position 6. By reacting with Kogyo Kogyo Co., Ltd., it is possible to easily detect the portion subjected to desulfurization. Therefore, FITC-labeled 2-B-6 and 3-B-3 antibodies can be reacted on the plate before and after mixed culture, and changes in fluorescence values can be easily detected. A compound with increased fluorescence intensity before and after the reaction can be determined to be a substance that further promotes desulfation, and can be easily identified as a novel therapeutic candidate compound that satisfies this concept.

 [0135] As another embodiment of the screening method of the present invention, there may be mentioned a method comprising a step of selecting a substance having a sulfation inhibitory action on chondroitin sulfate proteodalycan. The above method of the present invention also has the following process power, for example.

( _a ) A step in which a test compound is contacted with a substance or a cell extract expressing chondroitin sulfate proteodarican or a part thereof, or a substance group including an enzyme, a substrate, or the like that constitutes a sulfated process of chondroitin sulfate proteodarican.

 (b) a step of measuring sulfate activity of chondroitin sulfate proteoglycan in the cell, cell extract or substance group

 (c) A step of selecting a compound that decreases the activity compared to the case where the test compound is not contacted!

 [0136] In the above method, first, a test substance is brought into contact with chondroitin sulfate proteodarican or a part thereof.

[0137] Next, in this method, the amount of chondroitin sulfate proteodarican or a part thereof that has received sulfate is measured. The measurement can be performed by methods known to those skilled in the art. For example, chondroitin sulfate proteodarican or some of its sulfate Detection can be carried out by measuring the amount of label using a labeled compound or antibody that binds. Moreover, it can also detect using a chromatography method, a mass spectrometry, etc.

 [0138] In this method, a compound that reduces the abundance of the chondroitin sulfate proteodarican or a part thereof as compared with the case (control) when the test compound is not contacted with Select. The compound that lowers becomes a drug for the treatment of insulin resistance.

[0139] A simple example of a method and a specific example that can be evaluated (measured) as to whether or not the test compound has the activity of (d) sulfate inhibition is described below.

[0140] Aspects of the screening method relating to the above-mentioned (d) chondroitin sulfate proteodarican sulfate inhibitory action:

The cells and cell lines that promote the sulfation of chondroitin sulfate are the same as the cells and cell lines described in ( _c ) above. Various test compounds are mixed in the process of culturing such a cell line for a certain period of time, and the degree of sulfate before and after the culture is measured, for example, an antibody (clone; LY111 ) And antibodies that detect 6-position sulfation (clone; MC21C, also available from Seikagaku Corporation). Fluorescence-labeled antibodies may be used to compare fluorescence values before and after culture. Similarly to (c) above, detection methods using 2-B-6 and 3-B-3 antibodies may be performed before and after culture. Also good. Compounds that suppress the increase in sulfation after cell culture (LY111 and MC21C increase in fluorescence value!), Or progress of desulfation after cell culture (2-B-6 and 3-B-3 fluorescence values) A compound that promotes increased calorie) can be easily determined as a therapeutic candidate compound that satisfies this concept.

 [0141] Furthermore, more selectively, for example, a cell line in which a gene for a sulfotransferase such as C4ST-1 or C6ST-1 is introduced into CHO cells or L cells by a well-known method and is expressed constantly. Can be created. By using such a cell line to which a sulfate group is constantly added, it is possible to more clearly determine a treatment candidate compound.

[0142] Another preferred embodiment of the present invention is a compound that decreases the expression level of the chondroitin sulfate proteodarican core protein, the synthase, the desulfase inhibitor protein, or the sulfotransferase gene of the present invention, A compound that increases the expression level of the chondroitin sulfate proteoglycan degradation-promoting enzyme or desulfating enzyme gene A method for screening an insulin resistance inhibitor comprising the following steps (a) to (d).

( _a ) A test compound is brought into contact with a cell expressing a gene encoding a chondroitin sulfate proteodlican core protein, a synthetic enzyme, a desulfase inhibitor protein, a sulfotransferase, a degradation promoting enzyme, or a desulfase enzyme. Process

 (b) measuring the gene expression level in the cell

 (c) When the expression level is measured in the absence of the test compound!

 (d) When the gene is a chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, or sulfotransferase, the expression level of the gene is reduced compared to the control. When the compound or the gene is a chondroitin sulfate proteodarican degradation-promoting enzyme or a desulfurization enzyme, a step of selecting a compound having an increased expression level of the gene compared to the control

[0143] In the above method, first, a gene encoding a chondroitin sulfate proteodalycan core protein, a synthetic enzyme, a desulfurase inhibitor protein, a sulfotransferase, a degradation promoting enzyme, or a desulfurase is selected. A test compound is brought into contact with the cells to be expressed.

[0144] Next, in this method, the expression level of the gene encoding chondroitin sulfate proteodlican core protein, synthetic enzyme, desulfase inhibitor protein, sulfate transferase, degradation promoting enzyme, or desulfase is measured. To do. Here, “gene expression” includes both transcription and translation. The gene expression level can be measured by methods known to those skilled in the art.

[0145] For example, cellular force mRNA that expresses any of the above proteins is extracted according to a standard method, and Northern hybridization method, RT-PCR method, DNA array method, etc. using this mRNA as a cage are performed. Thus, the amount of transcription of the gene can be measured. Also

By collecting protein fractions from cells expressing genes encoding any of the above proteins, and detecting the expression of any of the above proteins by electrophoresis such as SDS-PAGE, You can also measure the amount of translation. Further, by performing Western blotting using an antibody against any of the above-mentioned proteins, the protein can be obtained. It is also possible to measure the amount of translation of a gene by detecting the expression of the protein. The antibody used for detecting the protein is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used.

 [0146] In this method, the expression level of the gene is compared with the case where the test compound is not brought into contact with the test compound!

 [0147] In this method, if the gene is a chondroitin sulfate proteodarican core protein, a synthase, a desulfase inhibitor protein, or a sulfotransferase, then the expression level of the gene is a control. Select a compound that is reduced (suppressed) compared to. A compound that decreases (suppresses) becomes a drug for suppressing insulin resistance or a candidate compound for treating insulin resistance.

 [0148] In addition, when the gene is a chondroitin sulfate proteodarican degradation-promoting enzyme or a desulfurization enzyme, the expression level of the gene is increased (enhanced) compared to the control. Select. Compounds that increase (enhance) become drugs for the inhibition of insulin resistance or candidate compounds for the treatment of insulin resistance.

 [0149] In addition, as one aspect of the screening method of the present invention, the expression level of the chondroitin sulfate proteodarican core protein, synthetic enzyme, desulfase inhibitor protein, or sulfotransferase gene of the present invention is reduced. This is a method of selecting a compound or a compound that increases the expression level of a chondroitin sulfate proteodarican degradation-promoting enzyme or desulfating enzyme gene using the expression of a reporter gene as an index. The method of the present invention includes, for example, the following steps (a) to (d).

( _a ) The transcriptional regulatory region of a gene encoding a chondroitin sulfate proteodarican core protein, a synthetic enzyme, a desulfase inhibitor protein, a sulfotransferase, a degradation-promoting enzyme, or a desulfase enzyme and the reporter gene are functional. A step of contacting a test compound with a cell or cell extract containing DNA having a bound structure

 (b) measuring the expression level of the reporter gene

 (c) Do not let the test compound come into contact!

(d) the reporter gene is a core protein of chondroitin sulfate proteodalycan, In the case of functionally binding to a synthase, a desulfurase inhibitor protein, or a sulfotransferase, a compound in which the expression level of the reporter gene is decreased compared to a control, the reporter gene is A step of selecting a compound in which the expression level of the reporter gene is increased compared to the control when functionally bound to an enzyme that promotes the degradation of chondroitin sulfate proteodarican or a desulfurization enzyme

 [0150] In this method! First of all, transcriptional regulation of a gene encoding chondroitin sulfate proteodarican core protein, synthetic enzyme, desulfase inhibitor protein, sulfotransferase, degradation promoting enzyme, or desulfurase oxidase A test compound is brought into contact with a cell or cell extract containing DNA having a structure in which a region and a reporter gene are functionally linked.

 [0151] Here, "functionally linked" refers to a gene encoding chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, sulfotransferase, degradation promoting enzyme, or desulfase So that transcription factor binds to the transcriptional regulatory region of the protein and induces the expression of the reporter gene, chondroitin sulfate proteodalycan core protein, synthase, desulfase inhibitor protein, sulfate transferase, accelerated degradation It means that a transcriptional regulatory region of an enzyme or a gene encoding a desulfating enzyme is linked to a reporter gene. Therefore, even when the reporter gene is linked to other genes and forms a fusion protein with other gene products, chondroitin sulfate proteodlican core protein, synthase, desulfase inhibitor protein, sulfate If the expression of the fusion protein is induced by binding of a transcription factor to the transcriptional regulatory region of a gene encoding a transferase, a degradation promoting enzyme, or a desulfating enzyme, the above-mentioned "functionally bound" Is included. Based on the cDNA base sequence of the gene encoding chondroitin sulfate proteodalican core protein, synthase, desulfase inhibitor protein, sulfate transferase, degradation promoting enzyme, or desulfase, The transcriptional regulatory region of the gene encoding chondroitin sulfate proteodlicancoprotein, synthetic enzyme, desulfase inhibitor protein, sulfotransferase, degradation-promoting enzyme, or desulfurization enzyme present in It is possible to obtain.

[0152] The reporter gene used in the present method is particularly restricted if its expression is detectable. Examples include CAT gene, lacZ gene, luciferase gene, and GFP gene. “Chondroitin sulfate proteodalycan core protein, synthase

A cell containing DNA having a structure in which a transcriptional regulatory region of a gene encoding a desulfase inhibitor protein, a sulfotransferase, a degradation promoting enzyme, or a gene encoding a desulfase enzyme and a reporter gene are functionally linked, for example, Examples include cells into which a vector having such a structure has been introduced. Such vectors can be prepared by methods well known to those skilled in the art. Introduction of the vector into the cells can be performed by a general method such as a calcium phosphate precipitation method, an electric pulse perforation method, a lipofussion method, a microinjection method, or the like. “A structure in which the transcriptional regulatory region of a gene encoding a chondroitin sulfate proteodlican core protein, synthase, desulfase inhibitor protein, sulfate transferase, degradation-promoting enzyme, or desulfase is functionally linked to a reporter gene The “cell containing DNA having a” includes a cell in which the structure is inserted into a chromosome. The DNA structure can be inserted into the chromosome by a method generally used by those skilled in the art, for example, a gene introduction method utilizing homologous recombination.

 [0153] "Transcriptional regulatory region of a gene encoding a chondroitin sulfate proteodarican core protein, a synthetic enzyme, a desulfurase inhibitor protein, a sulfotransferase, a degradation promoting enzyme, or a desulfase, and a reporter gene are functional. The cell extract containing DNA having a structure bound to is, for example, a cell extract contained in a commercially available in vitro transcription / translation kit, chondroitin sulfate proteolycan core protein, synthase, desulfate enzyme. For example, a DNA containing a structure in which a transcriptional regulatory region of a gene encoding a repressor protein, a sulfotransferase, a degradation promoting enzyme, or a desulfating enzyme and a reporter gene are functionally linked is included. it can.

[0154] Here, "contact" refers to "transcriptional regulation of a gene encoding chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, sulfotransferase, degradation-promoting enzyme, or desulfuric acid enzyme. A test compound is added to the culture solution of `` cells containing DNA having a structure in which a region and a reporter gene are functionally linked '', or a test compound is added to the above-described commercially available cell extract containing the DNA Can be done. When the test compound is a protein, for example, a DNA solid that expresses the protein It is also possible to carry out the introduction by introducing a cell into the cell.

 [0155] In this method, the expression level of the reporter gene is measured in the following manner. The expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of the reporter gene. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting the chloramfecole acetylene by the gene product. When the reporter gene is the lac Z gene, by detecting the color development of the dye compound by the catalytic action of the gene expression product, and when it is a luciferase gene, the fluorescent compound by the catalytic action of the gene expression product In the case of the GFP gene, the expression level of the reporter gene can be measured by detecting the fluorescence of the GFP protein.

 [0156] In this method, in the following, the measured expression level of the reporter gene is compared with that measured in the absence of the test compound (control).

 [0157] In this method, the reporter gene is then functionally linked to a chondroitin sulfate proteodarican core protein, a synthase, a desulfase inhibitor protein, or a gene encoding a sulfate transferase. Decrease (suppress) the expression level of the reporter gene compared to the control! / Select the compound to speak. A compound that lowers (suppresses) becomes a drug for suppressing insulin resistance or a candidate compound for treating insulin resistance.

 [0158] In addition, when the reporter gene is functionally linked to a chondroitin sulfate proteodarican degradation-promoting enzyme or a desulfurization enzyme, the expression level of the reporter gene is higher than that of the control. Increase (enhance) the compound! A compound that increases (intensifies) becomes a drug for suppressing insulin resistance or a candidate compound for treating insulin resistance.

 [0159] The insulin resistance inhibitor found in the screening method of the present invention is preferably for the treatment or prevention of an insulin resistance disease.

[0160] The present invention also provides a kit containing various drugs, reagents and the like used for carrying out the screening method of the present invention. [0161] The kit of the present invention can be appropriately selected from, for example, the above-mentioned various reagents of the present invention according to the screening method to be performed. For example, the kit of the present invention can comprise the chondroitin sulfate proteodarican of the present invention as a constituent element. The kit of the present invention can contain Sarako, various reagents and containers used in the method of the present invention. For example, an anti-chondroitin sulfate proteodarican antibody, a probe, various reaction reagents, cells, a culture solution, a control sample, a buffer solution, instructions describing how to use and the like can be appropriately included.

 [0162] In a preferred embodiment of the present invention, there is a method for screening an insulin resistance inhibitor, which comprises the step of detecting whether the production or accumulation of chondroitin sulfate proteodarican is inhibited. Therefore, in the screening method, for example, a probe for a gene encoding the core protein of chondroitin sulfate proteodarican that can be used for detection of chondroitin sulfate proteodarican, or a primer for amplifying an arbitrary region of the gene Oligonucleotides or antibodies that recognize chondroitin sulfate proteodarican (anti-chondroitin sulfate proteodarican antibody)

And can be included in the components of the insulin resistance inhibitor screening kit of the present invention.

 [0163] The oligonucleotide specifically hybridizes, for example, to the DNA of the Versican core protein gene of the present invention. As used herein, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambu Norec et al., Molecular Cloning. Cold Spring Harbor Laboratory Press, New York, In the USA, 2nd edition, 1989), which means that no significant cross-hybridization occurs with DNA encoding other proteins. If specific hybridization is possible, the oligonucleotide does not need to be completely complementary to the nucleotide sequence of the Versican core protein gene of the present invention! /.

In the present invention, hybridization conditions include, for example, “2 X SSC, 0.1% SDS, 50 ° C.”, “2 X SSC, 0.1% SDS, 42 ° C.”, “1 X SSC” , 0.1% SDS, 37 ° C '' and more stringent conditions as `` 2 X SSC, 0.1% SDS, 65 ° C '', `` 0.5 X SSC, 0.1% SDS, 42 ° C '' and `` 0.2 X SSC '' , 0.1% SDS, 65 ° C. ”. More specifically, Rapid As a method using -hyb buffer (Amersham Life Science), after pre-hybridization at 68 ° C for 30 minutes or more, add a probe and keep at 68 ° C for 1 hour or more to form a hybrid. Then wash 3 times for 20 minutes at room temperature in 2 X SSC, 0.1% SDS, followed by 3 times 20 minutes at 37 ° C in 1 X SSC, 0.1% SDS, and finally 1 X SSC. Can be washed twice for 20 minutes at 50 ° C in 0.1% SDS. In addition, for example, in Prehybridization Solution (CLONTECH), after prehybridization at 55 ° C for 30 minutes or more, add a labeled probe and incubate at 37-55 ° C for 1 hour or more. It is also possible to wash 3 times for 20 minutes at room temperature in 2 X SSC and 0.1% SDS, and once for 20 minutes at 37 ° C in 1 X SSC and 0.1% SDS. Here, for example, by setting the temperature at the time of pre-hybridization, hybridization, or the second washing to be higher, more stringent conditions can be obtained. For example, the temperature of the prehybridization and noble hybridization can be set to 60 ° C, and the stringent condition can be set to 68 ° C. Those skilled in the art will consider the conditions such as the salt concentration and temperature of the buffer, as well as other conditions such as the probe concentration, probe length, probe base sequence composition, and reaction time. Can be set.

 [0165] The oligonucleotide can be used as a probe or primer in the above-described screening kit of the present invention. When the oligonucleotide is used as a primer, the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least a part of the DNA of the gene of the present invention described above, for example, the force described in SEQ ID NO: 69 or 70!

 [0166] The present invention also provides a method for treating or preventing an insulin resistant disease, which comprises the step of administering the agent of the present invention to an individual (eg, a patient).

 [0167] The subject of the prevention or treatment method of the present invention is not particularly limited as long as it is an organism that can develop an insulin resistance disease, but is preferably a human.

[0168] Administration to an individual can be generally performed by methods known to those skilled in the art, such as intraarterial injection, intravenous injection, and subcutaneous injection. The dose varies depending on the weight and age of the patient, the administration method, etc., but a person skilled in the art (such as a doctor, veterinarian, pharmacist, etc.) can appropriately select an appropriate dose. [0169] Furthermore, the present invention relates to the use of the agent of the present invention in the manufacture of an insulin resistance inhibitor.

 [0170] It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

 Example

 [0171] Of course, there are no reports of actual clinical treatments that controlled the accumulation of these proteodaricans and observed pathological changes, but this time, as an example of this, Versicaiu core protein containing a core protein that is one of the chondroitin sulfate proteodaricans One of the enzymes that cleave the helical side chain when stretched. FcoChondroitin D—N—acetylgalactosamine— 4—0—sulfotransferase 1, Chondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 2, Chondroitin D—N—acetylgalactosamine— Examples are given focusing on 4-0-sulfotransferase 3 (C4ST-1, C4ST-2, C4ST-3). Various studies have been conducted so far, and there is no example regarding the application of Versican etc. to the power ^ type diabetes model (NIDD M), and it is expected as a therapeutic Z preventive drug by a new mechanism.

 [0172] Hereinafter, the present invention will be described more specifically with reference to Examples. However, the technical scope of the present invention is not limited to these examples.

 [0173] Experiment l: Streptozotodn-induced C57BL / 6.TcL Type 2 diabetes (NIDDM) model mice treated with Versican (siRNA), body weight, blood glucose level change, Versican gene expression, tissue level evaluation, examination

 In this example, a type 2 diabetes model was prepared by Streptozotocin administration of C57BL / 6JcL mice (female, manufactured by CLEA Japan, Inc.) on the second day after birth. Versican siRNA treatment caused body weight fluctuation, blood glucose level fluctuation, and gene expression by siRNA administration. investigated.

 First, the sample preparation was performed as follows.

[0174] Example 1 StreDtozotocin-induced C57BL / 6TcL Type 2 diabetes (NIDDM) model mouse body weight perturbation in Versican (siRNA) treatment

 14th day of pregnancy C57BL / 6JcL mice (CLEA Japan, Inc.) are bred and given birth, 2 days after birth

C57BL / 6JcL female mice (CLEA Japan), Streptozocin 10 mg / mL (SIGMA) 20 μL / head injected subcutaneously, CE-2 (CLEA Japan) feed until 4 weeks of age, Sterile water After 4 weeks of age, they were fed a High Fat Diet diet (manufactured by CLEA Japan, Inc.) and sterilized water for 2 weeks. In 2 weeks, siRNA Versican 1 μg (GeneWorld) mixed with 1% Atelocollagen (Koken) siRNA medium once / week 200 L intraperitoneally (lshot / week) The treatment was performed twice (2 weeks). On the 14th day of the experiment, BrdU 5 mg / mL (manufactured by ZyMED Laborat ory Inc.) was treated with 100 L in the tail vein, and after 1 hour, dissection was performed, and the liver, spleen, kidney, testis, ovary, and muscle were removed. Samples for immunostaining and gene expression analysis were obtained. The mouse body weight was measured over time until the day of dissection in the Control group and Versican siRNA-treated group, and is shown in FIG. The vertical axis represents body weight (g) and the horizontal axis represents the experimental period (days), and comparison was made between the Control group (n = 6) and the Versican siRNA-treated group (n = 6). From Fig. 1, compared with the Control group, a tendency to suppress weight gain was observed in the Versican siRNA-treated group.

 [0175] Example 2 StreDtozotocin induction C57BL / 6TcL type 2 diabetes mellitus (NIDDM) model mice in Versican (siRNA) treatment rfn.

 Human crystalline insulin (0.75 U / kg) was administered intraperitoneally as an insulin-tolerance test on day anatomy, and blood glucose levels at 0 minutes, 15 minutes, and 60 minutes were measured with a Daltest Ace blood glucose meter ( Measurement was carried out using Bombyx Pharmaceuticals Co., Ltd.) for evaluation. Figure 2 shows the changes in blood glucose level after 0, 15, and 60 minutes of intraperitoneal administration of human crystalline insulin (0.75 U / kg) in the Control group and Versican siRNA treatment group after Versican siRNA treatment. The vertical axis represents the blood glucose level (mg / dL), and the horizontal axis represents the insulin tolerance test, 0 minutes, 15 minutes, and 60 minutes after. Fig. 2 shows that no significant change was observed between the Control group and Versican siRNA treatment group at 0 and 15 minutes, and a significant change between the Control group and Versican siRNA treatment group after 60 minutes. Was recognized.

 [0176] Example 3 Evaluation and examination of expression level of Versican gene in Versican (siRNA) treatment with Streptozotocin-induced C57BL / 6.TcL type 2 diabetes (NIDDM) model mouse

Streptozocin-induced C57BL / 6JcL mice, 50 mg of organ (spleen) isolated from females, 1 mL of RNA-Bee (TEL-TEST) is added and electric homogenizer (DIGITAL HO MOGENIZER, ASONE) After grinding, chloroform 200 L (manufactured by Sigma Aldrich Japan) is gently mixed, then ice-cooled for about 5 minutes, and centrifuged at 12,000 rpm at 4 ° C for 15 minutes. Centrifoge 5417R, eppendorf The product was centrifuged. After centrifugation Transfer 500 L of the supernatant to another Eppendorf tube, add 500 μL of isoprop annol (Sigma Aldrich Japan) equivalent to the supernatant, mix, and then prepare 1 μL of glycogen (Invitrogen). And ice-cooled for 15 minutes. Centrifuge for 15 minutes at 12,000 rpm and 4 ° C after 15 minutes with ice cooling, and then wash the RNA precipitate obtained by washing 3 times with 75% Ethanol 1000 μL (Sigma Aldrich Japan) for 30 minutes. ~ After 1 hour of natural drying, dissolve in Otsuka Distilled Water 50 (Otsuka Pharmaceutical Co., Ltd.), dilute 100 times with Otsuka Distilled Water (Otsuka Pharmaceutical Co., Ltd.), and then UV plate (Corning Coaster) The RNA concentration in the sample extracted with the plate reader (POWER Wave XS, manufactured by BIO-TEK) was calculated.

[0177] Next, the following procedure was performed to perform RT reaction (cDNA synthesis).

 The calculated RNA sample was adjusted to a concentration of 500 ng / 20 / ζ L, heated at 68 ° C. for 3 minutes with BL OCK INCUBATOR (manufactured by ASTEC), and cooled on ice for 10 minutes. Prepare in advance after cooling on ice! RT Pre Mix solution (Composition: 25 mM MgCl 18.64 μL (Invitrogen)), 5 X Buffer 20

 2

 μL (Invitrogen), 0.1 M DTT 6.6 μL (Invitrogen), 10 mM dNTP mix 10 μL (Invitrogen), RNase Inhibitor 2 μL (Invitrogen), MMLV Reverse trans cri ptase 1.2 μL (Invitrogen), Random primer 2 μL (Invitrogen), sterile distilled water 19.56 L (Otsuka distilled water: Otsuka Pharmaceutical Co., Ltd.) in 80 μL cache BLOCK INCUBATOR (ASTEC) Heat at 42 ° C for 1 hour. After 1 hour, heat at 99 ° C for 5 minutes with BLOCK INCUBATOR (ASTEC), then cool on ice to prepare 100 L of cDNA and synthesize it. Using the obtained cDNA, PCR reaction was performed with the following composition.

[0178] PCR Buffer 2 μL [Composition: 166 mM (NH 2) SO (Sigma Aldrich Japan), 670 mM T

 4 2 4

 ris pH8.8 (Invitrogen), 67 mM MgCl-6H O (Sigma Aldrich Japan), 100 m

 twenty two

M 2-mercaptoethanol] (WAKO)], 25 mM dNTP mix 0.8 L (Invitrogen), DMSO 0.6 L (Sigma Aldrich Japan), Primer Forward 0.2 L (GeneWorld), Primer Reverse 0.2 L ( GeneWorld Co., Ltd.), Otsuka distilled water 15.7 L (Otsuka Pharmaceutical Co., Ltd.), Taq polymerase 0.1 μL (Perkin Elmer Co., Ltd.), and cDNA 1 μL obtained from the above were mixed together. Authorized Themal Cycler (eppendorff soil) 94. C 45 second, 56 ° C. 45 second, 72 ° C. 60 second 35 cycles were reacted. After completion of the reaction, add 2 μL Loading Dye (Invitrogen) to the obtained PCR product and prepare 1.5% UltraPure Agarose (Invitrogen) Kenole. And then electrophoresis was performed at 100 V for 20 minutes using Mupid-2 plus (manufactured by ADVANCE). After electrophoresis, diluted 10,000 times with IX LoTE [Composition: 3 mM Tris—HCl (pH7.5) (Invitrogen), 0.2 mM EDTA (pH7.5) (Sigma Aldrich Japan)] Ethydium Bromide (Invitrogen) The product was shaken in a staining solution for 20 to 30 minutes, and gel expression was confirmed with EXILIM (manufactured by CASIO) installed in I-Scope WD (manufactured by AD VANCE) to confirm gene expression.

 [0179] Fig. 3 shows the gene expression results of GAPDH and Versican in the Control group and Versican siRNA treatment group by RT-PCR.

 [0180] The Versican siRNA and Primer (Forward, Reverse) (manufactured by GeneWorld) used here are shown below.

 [Primer 酉 己 歹 IJ]

 * GAPDH (GeneWorld)

 Forward: 5,-CTGCCAAGTATGACATCA -3, (SEQ ID NO: 67)

 Reverse: 5,-TACTCCTTGGAGGCCATGTAG -3, (SEQ ID NO: 68)

 * Versican (GeneWorld)

 Forward: 5,-GACGACTGTCTTGGTGG-3, (SEQ ID NO: 69)

 Reverse: 5,-ATATCCAAACAAGCCTG-3, (SEQ ID NO: 70)

 [Versican siRNA cocktail sequence] (GenBank accession numoer BC09b495)

 (GeneWorld)

 5 '-ATGAAAGGCATCTTATGGATGTGCTCA-3' (SEQ ID NO: 71)

 5,-GGCAGCCACCAGCAGGTACACTCT-3, (SEQ ID NO: 72)

 5,-CTGCTCAACAGGCTTGTTTGGATAT-3 (SEQ ID NO: 73)

 [0181] From Fig. 3, expression of GAPDH, which is a positive control, was observed by RT-PCR in both the Control group and the Versica n (siRNA) treatment group. Furthermore, in Versican, expression was decreased in the Versic an (siRNA) treatment group compared to the Control group, and knockdown of the Versican gene was confirmed by administration of Atellocollagen vehicle Versican siRNA.

[0182] Example 4 Streptozotocin-induced C57BL / 6.TcL Evaluation and examination of spleen tissue level by immunostaining (APP, Insulin) in Versican (siRNA) treatment with type 2 diabetes (NIDDM) model mice The obtained tissue sample sections were stained with anti-Amyloid precursor protein antibody and anti-insulin antibody to evaluate and examine the expression at the tissue level. Fig. 4 shows tissue images of the Control group and Versic an siRNA treatment group.

 [0183] From Fig. 4, it was confirmed that the immunohistochemical staining was suppressed in the Versican siRNA-treated group as compared to the Amyloid precursor protein deposition force control group. As a result of staining with the anti-insulin antibody, it was observed that the expression of insulin-positive β cells in the spleen was increased in the Versican siRNA-treated group compared to the Control group.

 [0184] Example 5 Streptozotocin-induced C57BL / 6.TcL Evaluation of spleen tissue level by immunostaining (CS56) in Versican (siRNA) treatment using type 2 diabetes (NIDDM) model mice CS56 ( Staining was performed using an antibody that stains chondroitin sulfate proteodarican itself, and the expression at the tissue level was evaluated and examined. Fig. 5 shows the yarn and woven images of the Control group and Versican siRNA treatment group.

 [0185] From FIG. 5, as a result of staining with CS56 (an antibody that stains chondroitin sulfate proteodarican itself), deposits of chondroitin sulfate proteodarican (CS56) were strongly observed in the Control group, whereas Versican It was observed that chondroitin sulfate proteoglycan (CS56) deposition was suppressed in the siRNA-treated group.

 [0186] Experiment 2: StreDtozocin-induced C57ST / 6 (Chond roitin D—N—acetylgalactosamine—4—0—sulfotransferase l) siRNA, C4a f ~ 2 (Cnondroit in D—N—acetylgalactosamine— 4— u-sulfotransferase 2) siRNA, C4a f ~ 3 (Chondroitin DN-acetylgalactosamine-4-0-sulfotransferase 3) Body weight, blood glucose level perturbation, gene expression, evaluation of tissue level in siRNA treatment,

[0187] In this example, a C2BL-1 (Chondroitin DN-acetylgalactosam ine— 4—1) model was prepared by Streptozotocin administration of C57BL / 6JcL mice (female, manufactured by CLEA Japan, Inc.) on the second day after birth. O-sulfotransferase 1) siRNA ^ and 4ST-2 (and then honaroitin D—N—acetylgalactosamine — 4— O—sulfotransferase 2) siRNA, C4ST—3 (Chondroitin D—N—acetylgalactosamine— 4— O-sulfotransferase 3) We examined body weight fluctuation, blood sugar level fluctuation, and gene expression by siRNA administration. First, the sample preparation was performed as follows.

 Example 6 C4ST-1 (Chondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 1) siRNA, Streptozodn-induced C57BL / 6.TcL NIDDM model mouse 4ST—2 (Shon droitin D—N — Acetylgalactosamine— 4—0—sulfotransferase 2) siRNA, 4a Γ—3 (Cnondroi tin D—N—acetylgalactosamine—4—0—sulfotransferase 3) Body weight fluctuation during siRNA treatment Day 14 of pregnancy C57BL / 6JcL mice (CLEA Japan) C57BL / 6JcL mice females were injected subcutaneously into Streptozocin 10 mg / mL (SIGMA) 20 L / head, and CE-2 (CLEA Japan) until 4 weeks of age. Feed) and sterilized water were bred for 4 weeks and then fed with High Fat Diet (CLEA Japan) and sterilized water for 2 weeks. In the second week, siRNA and 4ST-1 (and nondroitin D—N—acetylgalactosamine—4—〇—sulfotransferase 1), then 4 ST—2 (Chondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 2), C4ST—3 (Ch ondroitin D—N—acetylgalactosamine—4-0—sulfotransferase 3) 1 μg (GeneWorld) mixed with 1% Atelocollagen (Koken), siRNA medium, once / week 200 Treatment was performed twice (2 weeks) by intraperitoneal administration of μ L (once / week). On the 14th day of the experiment, BrdU 5 mg / mL (manufactured by ZyMED Laboratory. Inc.) 100 L was administered into the tail vein, and after 1 hour, dissection was performed, and the liver, spleen, kidney, testis, ovary, and muscle were removed, Samples for immunostaining and gene expression analysis were obtained. Regarding fluctuations in body weight, measurements were performed over time for 14 days during the period of the examples. Figure 6 shows the results. The vertical axis shows body weight (g) and the horizontal axis shows days of measurement (days). As shown in FIG. 6, the C4ST-1 siRNA-treated group, the C4ST-2 siRNA-treated group, and the C4S T-3 siRNA-treated group showed a tendency to suppress weight gain after the 12th day of treatment.

 Example 7 Streptozocin-induced C57BL / 6.TcL NIDDM model mouse C4ST- Chondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 1) siRNA, 4ST—2 (and hon droitin D—N—acetylgalactosamine) — 4—0—sulfotransferase 2) siRNA, 4a Γ—3 (Cnondroi tin D—N—acetylgalactosamine—4—0—sulfotransferase 3) Changes in blood glucose level during siRNA treatment.

On the day before the dissection, human crystalline insulin (0.75 U / kg) was administered intraperitoneally as an insulin-tolerance test, and blood glucose levels at 0 minutes, 15 minutes, and 60 minutes were measured. Measurement was carried out using Yakuhin Co., Ltd. and evaluation was performed. 0 minutes after siRNA treatment Figure 7 shows the fluctuations in blood glucose levels after 15 minutes and 60 minutes. The vertical axis shows blood glucose level (mg / dL), and the horizontal axis shows 0 min, 15 min, and 60 min after insulin tolerance test. From Fig. 7, compared with the Control group, there was no significant decrease in blood glucose level after 0 min, 15 min after treatment in the C4ST-1 siRNA treatment group, C4ST-2 siRNA treatment group, and C4ST-3 siRNA treatment group. Unrecognized force 60 minutes later, a significant decrease in blood glucose level was observed in each of the C4ST-1 siRNA-treated group, the C4ST-2 siRNA-treated group, and the C4ST-3 siRNA-treated group.

 Example 8 Streptozodn-induced C57BL / 6.TcL NIDDM model mouse C4ST-1 (C hondroitin D—N—acetylgalactosamine—4-0—sulfotransferase 1) siRNA, 4ST—2 (and hon droitin D—N — Acetylgalactosamine— 4—0—sulfotransferase 2) siRNA, 4a Γ—3 (Cnondroi tin D—N—acetylgalactosamine—4—0—sulfotransferase 3) izs child in siRNA treatment ¾ Evaluation of current level

 Streptozocin-induced C57BL / 6JcL mice, 50 mg of organ (spleen) isolated from females, 1 mL of RNA-Bee (TEL-TEST) is added and electric homogenizer (DIGITAL HO MOGENIZER, ASONE) After grinding, chloroform 200 L (manufactured by Sigma Aldrich Japan) is gently mixed, then ice-cooled for about 5 minutes, and centrifuged at 12,000 rpm at 4 ° C for 15 minutes. Centrifoge 5417R, eppendorf The product was centrifuged. Transfer 500 L of the supernatant after centrifugation to another Eppendorf tube, add isoprop annol 500 μL (Sigma Aldrich Japan) equivalent to the supernatant, mix, and then 1 μL glycogen (Invitrogen) And cooled on ice for 15 minutes. Centrifuge for 15 minutes at 12,000 rpm, 4 ° C after 15 minutes with ice cooling, and then wash the RNA precipitate obtained by washing 3 times with 75% Ethanol 1000 μL (Sigma Aldrich Japan) for 30 minutes. ~ After 1 hour of natural drying, dissolve in Otsuka Distilled Water 50 (Otsuka Pharmaceutical Co., Ltd.), dilute 100 times with Otsuka Distilled Water (Otsuka Pharmaceutical Co., Ltd.), and then UV plate (Corning Coaster) The RNA concentration in the sample extracted with the plate reader (POWER Wave XS, manufactured by BIO-TEK) was calculated.

 [0191] For the RT reaction (cDNA synthesis), the calculated RNA sample was adjusted to a concentration of 500 ng / 20 μL, and the mixture was calored at 68 ° C for 3 minutes using BLOCK INCUBATOR (ASTEC). Warm and cool on ice for 10 minutes. RT Pre Mix solution (composition: 25 mM MgCl 18.64) prepared in advance after ice cooling

2 μL (Invitrogen), 5 X Buffer 2Q μL (Invitrogen), 0.1 M DTT 6.6 L (Invitro gen), 10 mM dNTP mix 10 L (Invitrogen), RNase Inhibitor 2 L (Invitrogen), MMLV Reverse transcriptase 1.2 μL (Invitrogen), Random primer 2 μL (Invitrogen) , Sterilized distilled water 19.56 μL (Otsuka distilled water: manufactured by Otsuka Pharmaceutical Co., Ltd.) with 80 μL Caloe BLOCK INCUBATOR (ASTEC), heated at 42 ° C for 1 hour, 1 hour later, BLOCK INCUBATOR After heating at 99 ° C for 5 minutes (manufactured by ASTEC) and cooling with ice, cDNA obtained by preparing cDNA 100 / zL to be obtained was subjected to PCR reaction with the following composition.

[0192] PCR Buffer 2 μL [Composition: 166 mM (NH 2) SO (Sigma Aldrich Japan), 670 mM T

 4 2 4

 ris pH8.8 (Invitrogen), 67 mM MgCl-6H O (Sigma Aldrich Japan), 100 mM

 twenty two

2-mercaptoethanol] (WAKO)], 25 mM dNTP mix 0.8 μL (Invitrogen), DM SO 0.6 μL (Sigma Aldrich Japan), Primer Forward 0.2 μL (GeneWorld), Primer Reverse 0.2 μL (GeneWorld), Otsuka distilled water 15.7 μL (Otsuka Pharmaceutical), Taq polymerase 0.1 L (Perkin Elmer), and 1 μL of cDNA obtained from above are mixed together to prepare an Authorized Themal. ycler, eppendori ^ earthenware) [from here 94. 45 seconc 5 ³ 45 second, 72 ° C 60 second 35cycle After completion of the reaction, add 2 μL Loading Dye (Invitrogen) to the resulting PCR product to prepare 1.5% UltraPure Agarose (Invitrogen Sento) Kenole, Mupid-2 plus (ADVANCE) From this, electrophoresis was performed at 100 V for 20 minutes. After electrophoresis, dilute 10,000 times with lX LoTE [Composition: 3 mM Tris—HCl (pH7.5) (Invitrogen), 0.2 mM EDTA (pH7.5) (Sigma Aldrich Japan)] Ethydium Bromide (Invitrogen After shaking for 20 to 30 minutes in a dye solution, gel photography was performed with EXILIM (CASIO) installed in I-Scope WD (ADVANCE) to confirm gene expression.

[0193] Primer (Forward, Reverse) used this time (GeneWorld)

 [Primer 酉 己 歹 lj]

 * GAPDH

 Forward: 5,-CTGCCAAGTATGACATCA -3, (SEQ ID NO: 67)

 Reverse: 5,-TACTCCTTGGAGGCCATGTAG -3, (SEQ ID NO: 68)

 * 4ST1 (and nondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 1)

 Forward: 5 — gtggatgaggaccacgaact— 3 (No. 74)

Reverse: 5, cttttcaagcggtggttgat— 3, (酉 ti 歹 U number: 75) * C4ST2 (Chondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 2)

 Forward: 5, -acctcctagacccacacacg-3, (selfie column number: 76)

 Reverse: 5-ggatgttggcaaaccagtct- «3 (Eye C Row ¾ · No .: i l)

 * 4ST »j (Nondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 3)

 Forward: 5-atgagcccttcaacgaacac-3 (Self number: 78)

 Reverse ： 5-tggtagaaggggctgatgtc-(eye cl sequence ¾ · No .: ί'9)

 * [C4ST-1 siRNA cocktail sequence]

4ST1 (and hondroitin D—N—acetylgalactosamine—4—〇—sulfotransferase 1)

(uenBank accession number NM— 021439)

T-3 '(SEQ ID NO: 80)

 5 '-CAACCTGAAGACCCTTAACCAGTACA-3' (SEQ ID NO: 81)

5, -GCATCCCAGAGATCAACCACCGCTTG-3, (SEQ ID NO: 82)

* [C4ST-2 siRNA cocktail sequence]

4ST2 (and hondroitin D—N—acetylgalactosamine—4—〇—sulfotransferase 2)

(uenBank accession number NM_021528J

 5 '-GCCAGGAGTGGGCCCAGCCCAGGGC -3, (SEQ ID NO: 83)

5 '-ATGACCAAGCCGCGGCTCTTCCGGCTG-3' (SEQ ID NO: 84)

5'-AGAGCCTGCTGGACCGGGGCAGCCCCTA-3, (SEQ ID NO: 85)

5, -GAGACCCCCTGGACATCCCCCGGGAACA-3, (SEQ ID NO: 86)

* [C4ST-3 siRNA cocktail sequence]

4ST »j (and hondroitin D—N—acetylgalactosamine—4—〇—sulfotransferase 3)

(uenBank accession number XM_J55798J

 5 '-ATGACTGTCGCCTGCCACGCGTGCCA -3, (SEQ ID NO: 87)

5 '-CAGCATGGGAAGACGCTCCTGTTGCA -3' (SEQ ID NO: 88)

5'-TCCAAGCGCAATCCCTGCGCACGAGGCG-3, (SEQ ID NO: 89)

5,-GCCTGGCCTGCTGCCCTCGCTGGCC-3 '(SEQ ID NO: 90)

The results are shown in FIG. The expression of GAPDH, which is a positive control, is controlled by RT-PCR. C4ST1 (Chondroitin D—N—acetylgalactosamine— 4—0—sulfotransferase 1), C4ST2 (Chondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 2), C4ST3 (Chondroit in D—N—acetylgalactosamine—4—0— C4ST1 (Chondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 1) siRNA, C4ST2 (Chondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 2) ) siRNA, C4ST3 (Chondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 3) Decreased expression was observed in siRNA treatment group, Atellocollagen medium C4ST1 (Chondroitin D-N-acetylylactactamine—4—0—sulfotransferase 1) siRNA 4ST2 (and nondroitin D—N—acetylgal actosamine—4—0—sulfotransferase 2) siRNA, C4ST3 (Chondroitin D—N—acetylgalacto samine- 4-0-sulfotransferase 3) C4ST1 (Chondroitin DN-acetylgal actosamine-) 4-0- sulfotransferase 1) C4ST2 (Chondroitin DN-acetylgalactosamine-4-0-sulfotransferase 2) and C4ST3 (Chondroitin DN-acetylgalactosamine-4-0-sulfotransferase 3) genes were confirmed to be knocked down.

Example 9 StreDtozodn Induction Case C57ST / 6 (Chondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 1) siRNA, 4ST—2 (and hon droitin D—N—acetylgalactosamine) in C57BL / 6TcL NIDDM model mice — 4—0— sulfotransferase 2) siRNA, shi 4a Γ— 3 (Cnondroi tin DN-acetylgalactosamine-4-0-sulfotransferase 3) Evaluation of tissue level (APR Insulin) using immune ¾ color in siRNA treatment

 The obtained tissue sample sections were stained with anti-Amyloid precursor protein antibody and anti-insulin antibody to evaluate and examine the expression at the tissue level. FIG. 9 shows tissue images of the Control group, the C4ST-1 siRNA treatment group, the C4ST-2 siRNA treatment group, and the C4ST-3 siRNA treatment group.

 [0196] From FIG. 9, it can be seen that the myelin precursor protein is deposited in the C4ST-1 siRNA treatment group, the C4ST-2 siRNA treatment group, and the C4ST-3 siRNA treatment group in comparison with the control group. Was confirmed to be suppressed. In addition, as a result of staining with anti-insulin antibody, the expression of insulin positive β cells in the spleen increased in the C4ST-1 siRNA treatment group, the C4ST-2 siRNA treatment group, and the C4ST-3 siRNA treatment group as compared with the Control group, It was observed that

Example 10 StreDtozotocin-induced C57BL / 6.TcL type 2 diabetes (NIDDM) model mouse 【Cydroitin D—N—acetylgalactosamine— 4—〇—sulfotransferase l) siRN A, C4ST-2 (Chondroitin D—N—acetylgalactosamine— 4—〇—sulfotransferase 2) siRNA, C4ST-3 (Chondroitin D —N— acetylgalactosamine— 4—〇—sulfotransferase 3) Evaluation of knee tissue level by immunostaining (esse) in siRNA treatment,

 The obtained tissue sample sections were stained with CS56 (an antibody that stains chondroitin sulfate proteodarican itself) to evaluate and examine the expression at the tissue level. FIG. 10 shows tissue images of the Control group, the C4ST-1 siRNA treatment group, the C4ST-2 siRNA treatment group, and the C4ST-3 siRNA treatment group.

 [0198] From Fig. 10, as a result of staining with CS56 (an antibody that stains chondroitin sulfate proteodarican itself), deposits of chondroitin sulfate proteodarican (CS56) were strongly observed in the Control group, whereas C4ST It was observed that the deposition of chondroitin sulfate proteodarican (CS56) was suppressed in the -1 siRNA-treated group, the C4ST-2 siRNA-treated group, and the C4ST-3 siRNA-treated group.

 Industrial applicability

[0199] As an example of examining the effect of accumulation of chondroitin sulfate proteoglycan (CSPG) according to the present invention, a side of a chondroitin sulfate proteoglycan containing a Versican core site sequence, which is one of the chondroitin sulfate proteoglycans. C4ST1 (Chondroitin D-N-acetylgala ctosamine- 4-O-sulfotransferase 1), C4¾ Γ2 (Cnondroitin DN-acetylgalactosamine- 4— O— sulfotransferase 2), C4ST3 ( Chondroitin D—N—acetylgalactosamine— 4—0— sulfot ransferase 3) siRNA suppresses chondroitin sulfate proteoglycan accumulation in splenic Langerhans islet j8 cells and suppresses insulin resistance to treat type 2 diabetes (NIDDM) Or it has an effect on prevention. The insulin resistance inhibitor according to the present invention is a chondroitin sulfate proteo around the splenic Langerin's Islet j8 cells whose Versican expression is suppressed by administration of Versica siRNA, C4ST-1 siRNA, C4ST-2 siRNA, C4ST-3 siRNA. Since it has an inhibitory effect on glycan accumulation, it is very useful in suppressing insulin resistance of splenic Langerhans islet β cells. Type 2 diabetes by administering an insulin resistance inhibitor having an inhibitory effect on chondroitin sulfate proteoglycan accumulation of the present invention (NIDDM) Disease treatment or prevention methods can be an excellent therapy useful for further improvement of patients' quality of life and medical care because the pathology can be effectively improved by an unprecedented mechanism of action and drug therapy. .

 All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

The scope of the claims  [I] An insulin resistance inhibitor comprising, as an active ingredient, a substance that inhibits the production or accumulation of chondroitin sulfate proteodarican.  [2] The drug according to claim 1, wherein the substance is a substance having a chondroitin sulfate proteodarican degradation promoting action. [3] The drug according to claim 1, wherein the substance is a substance having a chondroitin sulfate proteodarican synthesis inhibitory action. [4] The drug according to claim 1, wherein the substance is a substance having a chondroitin sulfate proteodarican desulfation effect. [5] The drug according to claim 1, wherein the substance is a substance having an inhibitory action on chondroitin sulfate proteodalycan sulfate. [6] The drug according to any one of claims 1 to 5, wherein production or accumulation of chondroitin sulfate proteodarican is inhibited in spleen β cells. [7] The drug according to any one of claims 1 to 6, which is used for treatment or prevention of an insulin resistant disease.  [8] The insulin resistant disease is an insulin resistant disease associated with metabolic syndrome. The drug according to claim 7. [9] The drug according to claim 7, wherein the drug is insulin-resistant illness insulin-independent (type 2) diabetes.  [10] A method for screening an insulin resistance inhibitor, comprising selecting a substance having an action of inhibiting the production or accumulation of chondroitin sulfate proteodarican from a test sample.  [II] The screening method according to claim 10, comprising a step of selecting a substance having the action described in any one of (a) to (d) below. ( _a ) Promoting the degradation of chondroitin sulfate proteodarican  (b) Inhibition of chondroitin sulfate proteodarican synthesis  (c) Desulfation effect of chondroitin sulfate proteodarican (d) Sulfate inhibitory action of chondroitin sulfate proteodarican 12. The screening method according to claim 10 or 11, wherein the insulin resistance inhibitor is for treatment or prevention of an insulin resistance disease.