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WO2006115047A1 - Gene dont l’expression change en fonction des troubles du diabete et son utilisation - Google Patents

Gene dont l’expression change en fonction des troubles du diabete et son utilisation Download PDF

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WO2006115047A1
WO2006115047A1 PCT/JP2006/307700 JP2006307700W WO2006115047A1 WO 2006115047 A1 WO2006115047 A1 WO 2006115047A1 JP 2006307700 W JP2006307700 W JP 2006307700W WO 2006115047 A1 WO2006115047 A1 WO 2006115047A1
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chromosome
reading frame
open reading
gene
type
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Shuichi Kaneko
Toshinari Takamura
Hirofumi Misu
Taro Yamashita
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Kanazawa University NUC
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Kanazawa University NUC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the use of genes that can be key molecules for the pathogenesis of sugar'lipid metabolism, diabetic complications, arteriosclerosis, etc., and the present invention further varies in expression in relation to the pathology of diabetes. Further, the present invention relates to the use of the gene for diagnosis or risk assessment of diabetes or obesity.
  • Type 2 diabetes 'obesity' continues to increase on a global scale and promotes arteriosclerosis such as retinal 'renal' nerve complications and ischemic heart disease, threatening human QOL and life.
  • the liver is an important organ that plays a central role in glucose and fat metabolism.
  • insulin resistance the action of insulin to suppress the release of sugar by hepatic strength is attenuated, and this phenomenon is called insulin resistance (see Non-Patent Documents 1 and 2).
  • Insulin resistance leads to hyperglycemia due to increased sugar release by liver strength and hyperlipidemia due to increased lipid production, and both promote arteriosclerosis.
  • the liver is the largest organ in the body of various physiologically active substances including angiogenic factors that lead to the risk of arteriosclerosis.
  • Non-Patent Document 1 Michael MD. Et al., Mol. Cell 6: 87-97, 2000
  • Non-Patent Document 2 Saltiel AR. Et al., Nature 414: 799-806, 2001
  • the present invention relates to a gene that can be a key molecule for producing a pathological condition such as sugar'lipid metabolism, diabetic complications, arteriosclerosis, and the sugar'lipid metabolism, diabetic complications, arteriosclerosis of the gene.
  • the method of use to elucidate the cause of pathological conditions such as is provided.
  • the present invention relates to a reagent for measuring the expression of a gene whose expression varies in relation to diabetes or obesity using a gene whose expression varies in relation to the pathological condition of diabetes, and diabetes using the reagent,
  • the purpose is to provide methods for detecting obesity, diabetic complications, arteriosclerosis, and the like.
  • the present inventor has constructed the world's largest liver-expressed gene database that exceeds the 600,000-expressed genes obtained from SAGE of normal liver and various liver diseases and DNA chip analysis so far, and analyzes liver disease. I have recommended.
  • a cDNA microarray originally developed in the process, the profile of the gene expressed in the liver of type 2 diabetic patients was analyzed, and it was thought that there was no abnormality in the conventional research method.
  • the expression of various known genes related to vascular complications such as angiogenic factors that are not only directly related to glucose metabolism was increased (Takamura T. et al. al., Diabetologia 47: 638-647, 2004). These known genes may shape the pathology of type 2 diabetes.
  • liver-derived secretory proteins may have unknown secreted proteins that shape the pathology of diabetes, including vascular complications, in livers with type 2 diabetes and obesity. . Therefore, this original research was further developed, and the gene produced by the liver of type 2 diabetic patients, including unknown genes with unknown functions, was analyzed using the serial analysis of gene expression method (hereinafter referred to as SAGE method).
  • SAGE method serial analysis of gene expression method
  • a gene whose expression is increased or attenuated in the liver tissue of a human type 2 diabetic or obese patient compared to the liver tissue of a normal human subject, which encodes a secreted protein In order to measure the expression of type 2 diabetes-related genes and Z or obesity-related genes containing nucleotides consisting of the base sequence of at least one selected gene or nucleotides including a partial sequence thereof Reagent.
  • nucleobindin 2 nucleobindin 2
  • matrix metalloproteinase 11 matrix metalloproteinase 11 (stromelysin 3)
  • DST Homo sapiens dystonin
  • glycosylphosphatidylinositol specific phospholipase Dl (32) glycosylphosphatidylinositol specific phospholipase Dl (32) GDF15 growth differentiation factor 15
  • ADAMTSl a disintegrin like and metalloprotease (reprolysin type) with thrombo spondin type 1 motif 1
  • a gene whose expression is up- or down-regulated in the liver tissue of a human type 2 diabetes patient is selected from the group consisting of the following genes whose up- or down-expression is correlated with HbAlc levels in human blood
  • matrix metalloproteinase 11 (stromelysin 3)
  • a gene whose expression is up- or down-regulated in the liver tissue of a human type 2 diabetes patient is at least 1 selected from the group consisting of the following genes whose up-regulation or attenuation is correlated with HOMA-R
  • Phospholipase A2 group VII (platelet-activating factor acetylhydrolase, plasma)
  • a gene whose expression is up- or down-regulated in the liver tissue of human type 2 diabetics is correlated with increased or decreased expression.
  • ADAMTSl a disintegrin like and metalloprotease (reprolysin type) with thrombo spondin type 1 motif 1
  • a gene whose expression is up- or down-regulated in liver thread and tissue of human obesity patients is selected from the group consisting of the following genes whose up-regulation or attenuation is correlated with BMI: A reagent for measuring the expression of genes related to type 2 diabetes and Z or obesity related genes of [1].
  • DST Homo sapiens dystonin
  • a reagent for measuring the expression of a type 2 diabetes-related gene and a Z- or obesity-related gene according to any one of [1] to [5], comprising at least 5 genes.
  • a nucleotide comprising a nucleotide sequence or a partial sequence thereof that also has a base sequence ability of a gene whose expression is increased or decreased in the liver tissue of a human type 2 diabetic patient compared to a liver tissue of a normal human subject
  • a nucleotide comprising a nucleotide sequence or a partial sequence thereof, which also has a base sequence ability of a gene whose expression is increased or decreased in the liver tissue of a human type 2 diabetic patient compared to the liver tissue of a normal human subject
  • the expression in a subject is measured, and if the expression of the gene is increased or decreased compared to a normal person, the subject is type 2 diabetes, How to determine that you have or are at risk of suffering from obesity, diabetic complications or arteriosclerosis.
  • matrix metalloproteinase 11 (stromelysin 3)
  • DST Homo sapiens dystonin
  • ADAMTSl a disintegrin like and metalloprotease (reprolysin type) with thrombo spondin type 1 motif 1
  • a gene whose expression is up- or down-regulated in the liver tissue of a human type 2 diabetes patient is a group consisting of the following genes whose up- or down-expression is correlated with HbAlc levels in human blood: The method of [9], which is a single gene.
  • matrix metalloproteinase 11 (stromelysin 3)
  • a gene whose expression is enhanced or attenuated in the liver tissue of a human type 2 diabetic patient is selected as a group force with the following gene strength in which increased or attenuated expression is correlated with HOMA-R: at least 1 selected The method of [9], which is one gene. [0018] (42) thrombospondin 1
  • Phospholipase A2 group VII (platelet-activating factor acetylhydrolase, plasma)
  • group VII platelet-activating factor acetylhydrolase, plasma
  • ADAMTSl a disintegrin like and metalloprotease (reprolysin type) with thrombo spondin type 1 motif 1
  • chromosome 5 open reading frame 13 (61) chromosome 6 open reading frame 79
  • a gene whose expression is enhanced or attenuated in the liver tissue of a human type 2 diabetes patient is at least one gene selected from the group consisting of the following genes whose expression is correlated with BMI: There is a method of [9].
  • DST Homo sapiens dystonin
  • a nucleotide comprising a nucleotide sequence or a partial sequence thereof, which also has a base sequence ability of a gene whose expression is increased or attenuated in the liver tissue of a human type 2 diabetic patient compared to the liver tissue of a normal human subject [9] to [14] !, either method.
  • a nucleotide comprising a nucleotide sequence or a partial sequence thereof that also has a base sequence ability of a gene whose expression is increased or attenuated in the liver tissue of a human type 2 diabetic patient compared to the liver tissue of a normal human subject [9] to [14], any of the methods including analyzing the expression of the gene by real-time PCR targeting at [9].
  • Type 2 diabetes, obesity, diabetic comprising a type 2 diabetes-related gene according to any one of [1] to [8] and a reagent for measuring the expression of Z or an obesity-related gene Kit for detection or risk assessment of complications and arteriosclerosis.
  • the gene group can be a key molecule for the pathogenesis of sugar and lipid metabolism, diabetic complications, arteriosclerosis, etc. By using this gene, sugar / lipid metabolism, diabetic complications, arteriosclerosis, etc. It is possible to elucidate the mechanism of the pathophysiology.
  • the gene whose expression changes in relation to the pathological condition of obesity or obesity of the present invention is a gene whose expression is different between the liver of a type 2 diabetes patient or obese patient and the liver of a normal person, and the liver of a normal person In comparison with the above, it is a gene whose expression in the liver of type 2 diabetic patients or obese patients is increased or decreased. Furthermore, the gene of the present invention is a gene encoding a secretory protein that is secreted extracellularly.
  • type 2 diabetes is a type of diabetes that develops due to a decrease in insulin secretion and a decrease in sensitivity to insulin.
  • a normal person means a person who does not exhibit type 2 diabetes, specifically, a person with normal insulin resistance in the liver or skeletal muscle, that is, a person with normal glucose tolerance.
  • blood HbAlc (hemoglobin Ale) concentration increases, and insulin resistance (insulin resistance in the liver and insulin resistance in skeletal muscle) power S is increased.
  • Insulin resistance in the liver can be determined by measuring HOMA-R (homeostasis model assessment), and the higher the value of HOMA-R, the stronger the insulin resistance in the liver.
  • Insulin resistance in skeletal muscle can be determined by measuring the glucose metabolic clearance rate (MCR). The larger the MCR value, the stronger the insulin resistance in skeletal muscle.
  • MCR glucose metabolic clearance rate
  • HOM AR values fasting blood glucose ⁇ / (11) fasting Insurin concentration (11 1?) (U / ml) / 405 indicated by (M atthews DR et al, Diabetologia 28:. 412-419, 1985), This value is around 1 for normal people.
  • the MCR value can be measured by, for example, the method described in DeFronzo RA et al., Am J Physiol 237: E214-223, 1979.
  • Obese patients are those whose BMI (Body Mass index) is high, and BMI is expressed as body weight (kg) Z height (m) 2 . If the BMI is 25 or greater, it is determined to be obese.
  • the gene whose expression changes in association with the pathological condition or obesity of type 2 diabetes of the present invention is a gene that is significantly correlated with any one of blood HbAlc level, HOMA-R, MCR and BMI.
  • a significant correlation is observed when, for example, the relationship between the measured values of blood HbAlc, HOM AR, MCR and BMI and the expression level of each gene is analyzed by linear correlation analysis. It means being done.
  • genes shown in Table 3 as genes that are significantly correlated with HbAlc levels in blood, and 29 genes shown in Table 4 as genes that are significantly correlated with BMI are significantly correlated with HOMA-R.
  • the 6 genes shown in Table 5 are recognized genes, and the 21 genes shown in Table 6 are examples of genes that have a significant correlation with MCR.
  • genes that have a significant correlation with the HbAlc value are found to have a significant correlation with the HbAlc disease-related gene group, genes that have a significant correlation with the HOMA-R, a HOMA-R disease-related gene group, and MCR.
  • Genes may be referred to as MCR pathology-related genes, and genes that are significantly correlated with BMI may be referred to as BMI pathology-related genes.
  • BMI pathology-related genes genes that are significantly correlated with BMI
  • the HbAlc disease-related gene group, the HOM A-R disease-related gene group, and the MCR disease-related gene group can be referred to as a type 2 diabetes-related gene group
  • the BMI disease-related gene group can be referred to as an obesity-related gene group. Since each gene group has some overlap, there are a total of 62 types of genes.
  • genes include genes whose functions have not been elucidated (in the column “Symbol” in Tables 3 to 6, c (n) ori (m) ((n) and (m) are integers)
  • a protein encoded by such a gene whose function is unknown is also known to be a secreted protein having a signal peptide from amino acid sequence analysis. was first found to be associated with diabetes and obesity.
  • SEQ ID NO: 1 PVR
  • SEQ ID NO: 2 LGALS8
  • SEQ ID NO: 3 FCN3
  • SEQ ID NO: 4 MMP24
  • SEQ ID NO: 5 MDK
  • No. 6 NUCB2
  • SEQ ID NO: 7 MMP11
  • SEQ ID NO: 8 GPX3
  • SEQ ID NO: 9 C14ori2
  • SEQ ID NO: 10 C 14orf4
  • SEQ ID NO: ll ClOorflO
  • SEQ ID NO: 12 C6orf74
  • Sequence It is represented by number 13 (C9or! 83) and SEQ ID NO: 14 (C8orfl).
  • SEQ ID NO: 15 BMP2
  • SEQ ID NO: 16 LECT2
  • SEQ ID NO: 17 ADFP
  • SEQ ID NO: 18 BPAG1
  • SEQ ID NO: 19 SEPINF1
  • SEQ ID NO: 20 GNAS
  • SEQ ID NO: 21 AHSG
  • SEQ ID NO: 22 CCL16
  • SEQ ID NO: 23 AFM
  • SEQ ID NO: 24 LTBP1
  • SEQ ID NO: 25 C4BPA
  • SEQ ID NO: 26 LYZ
  • SEQ ID NO: 4 MMP 24
  • SEQ ID NO: 27 PSAP
  • SEQ ID NO: 28 HRG
  • SEQ ID NO: 29 FST
  • SEQ ID NO: 30 GCC3
  • SEQ ID NO: 42 THBS1
  • SEQ ID NO: 43 CSS
  • SEQ ID NO: 44 PON1
  • SEQ ID NO: 45 DEFB1
  • SEQ ID NO: 46 MA2K2
  • PAG7 PAG7
  • nucleotide sequences of 21 genes shown in Table 6 are represented by SEQ ID NO: 36 (THBS1), SEQ ID NO: 43 (CTSS), SEQ ID NO: 48 (IGFBP1), SEQ ID NO: 49 (SULF2), SEQ ID NO: 50 (WNT5B).
  • SEQ ID NO: 51 (ADAMTS1), SEQ ID NO: 52 (SEPP1), SEQ ID NO: 53 (CHAD), SEQ ID NO: 54 (DCN), SEQ ID NO: 56 (LBP), SEQ ID NO: 4 (MMP24), SEQ ID NO: 57 (SPARCL1), SEQ ID NO: 58 (LRG1), SEQ ID NO: 59 (cHorflOO), SEQ ID NO: 35 (cl4orfl23), SEQ ID NO: 9 (C14orl2), SEQ ID NO: 60 (C18orfl0), SEQ ID NO: 37 (C5orfl3), SEQ ID NO: 61 (C6orf79), SEQ ID NO: It is represented by number 62 (C6ori80) and SEQ ID NO: 38 (C9orfl0).
  • the indication in parentheses indicates “Symbol” in the table.
  • Nucleotides used in the present invention include nucleotides containing the above sequences and fragments thereof.
  • the nucleotides used in the present invention also include nucleotides and fragments thereof that are hybridized under stringent conditions with the nucleotides shown in SEQ ID NOs.
  • Such nucleotides include, for example, a base sequence that is about 80% or more, preferably about 90% or more, more preferably about 95% or more on average in terms of the degree of homology with the above base sequence.
  • a nucleotide etc. can be mentioned. Hybridization is well known in the art, including the method described in Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987).
  • ⁇ stringent conditions '' are, for example, conditions of ⁇ 1XSSC, 0.1% SDS, 37 ° C '', and more severe conditions are ⁇ 0.5XSSC, 0.1% SDS, 42 ° C '' The more severe condition is “0.2XSSC, 0.1% SDS, 65 ° C”. As the hybridization conditions become more severe in this way, it can be expected to isolate a nucleotide having a high homology with the probe sequence.
  • the combinations of the above SSC, SDS and temperature conditions are exemplary, and those skilled in the art will recognize the above or other factors (eg, probe concentration, It is possible to achieve the same stringency as described above by appropriately combining the length of the probe and the reaction time of the hybridization.
  • some of the above genes (LGAL S8, FCN3, MDK, GNAS, LTBP1, FST, GPLD1) have a Norint, and the nucleotide of the present invention includes a nucleotide containing the nucleotide sequence of the Norint or a nucleotide thereof. Includes fragments.
  • nucleotide used in the present invention it is also possible to use a shift between a nucleotide comprising the sense strand of the above gene and a nucleotide comprising the antisense strand.
  • genes shown in Tables 3-6 those with a value of “DM (type 2 diabetic patients) / NGT (normal glucose tolerance patients) by SAGE” greater than 1 are pathological conditions of type 2 diabetes Or a gene whose expression is increased in relation to obesity, and a gene whose expression is attenuated in relation to the pathology of type 2 diabetes or obesity.
  • a gene with a higher value or a lower value can be said to be a gene having a stronger association with the pathology of type 2 diabetes or obesity. Therefore, among the genes listed in the table, genes with a value represented by “DM (type 2 diabetic patients) / NGT (normal glucose tolerance patients) by SAGE” in the table are 2 or more or 0.5 or less. It can be used more suitably.
  • This SAGE method is a method for determining a 10 base pair DNA sequence (Tag) that characterizes each of the cDNAs that have been prepared for gene expression and expressed in an arbitrary cell.
  • Tag 10 base pair DNA sequence
  • Ma the expression frequency (copy number) of each gene can be determined by calculating the ratio of all the obtained tag powers and individual tags.
  • genes can be key molecules for pathological conditions such as sugar-lipid metabolism, diabetic complications, and arteriosclerosis.
  • these genes are used to study glycolipid metabolism, elucidate the etiology of type 2 diabetes and obesity, detect (diagnose) type 2 diabetes, evaluate the risk of suffering from type 2 diabetes, and become obese It can be used for risk assessment and development of new treatments for diabetes and obesity.
  • these genes are closely related to insulin resistance, and can be used for determination of insulin resistance.
  • these genes are also associated with diabetic complications and can be used to detect (diagnose) diabetic complications or to determine the risk of suffering from diabetic complications.
  • diabetes, obesity, and insulin resistance are risk factors for arteriosclerosis, and these genes are also closely related to arteriosclerosis, which is associated with the detection (diagnosis) of arteriosclerosis and arteriosclerosis. It can also be used to determine risk. Examples of diabetic complications include diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic macroangiopathy, myocardial infarction, and stroke.
  • type 2 diabetes, obesity, diabetic complications or arteriosclerosis Even if it is possible to measure changes in the expression of genes related to the pathology of the disease and not detectably present the pathology, it may be determined that the patient is suffering from type 2 diabetes, diabetic complications or arteriosclerosis is there.
  • a condition is a condition that is likely to cause detection of type 2 diabetes, obesity, diabetic complications or arteriosclerotic pathology in the future, and this is a type 2 diabetes, obesity, diabetic complication. Can assess the risk of developing a pathology of atherosclerosis or arteriosclerosis.
  • At least one of the genes belonging to the HbAlc disease state-related gene group, the HOMA-R disease state-related gene group, the MCR disease state-related gene group, and the BMI disease state-related gene group is used.
  • At least one of the genes belonging to each of the HbAlc disease state-related gene group, the HOMA-R disease state-related gene group, the MCR disease state-related gene group, and the BMI disease state-related gene group preferably at least 2, more preferably 5 may be used, more preferably at least 10, more preferably all.
  • the BMI condition-related genes are predicted to be associated with obesity and can be used to assess the risk of becoming obese.
  • the degree of gene expression may be measured using nucleotides containing all or part of the base sequences of the above genes as probes or primers.
  • the level of gene expression can be measured by the Northern plot method, RT-PCR method, real-time PCR method, in situ hybridization method, microarray (microchip) method, etc. Any of the methods mentioned can be carried out by known methods.
  • the expression of a gene can be measured by using a nucleotide probe or primer that hybridizes to the mRNA as long as the amount of messenger RNA (mRNA) transcribed from a part of the gene is measured.
  • mRNA messenger RNA
  • the base length of the probe or primer is 10 to 50 bp, preferably 15 to 25 bp.
  • a liver biopsy sample, serum, plasma or the like may be used as a sample sample for evaluating whether or not there is a risk of suffering from sclerosis.
  • the expression of one or more genes belonging to the HbAlc disease-related gene group, the HOMA-R disease-related gene group, the MCR disease-related gene group, and the BMI disease-related gene group is increased or attenuated compared to normal individuals.
  • the subject can be diagnosed as having type 2 diabetes, obesity, diabetic complications or arteriosclerosis, and the subject has type 2 diabetes, hypertrophy, diabetic complications or arteries. Can be assessed as having a risk of suffering from sclerosis.
  • the quantification of the extent of the attenuation makes the subject more accurate, such as type 2 diabetes, obesity, Can be diagnosed as suffering from diabetic complications or arteriosclerosis and assessed that the subject is at risk of suffering from type 2 diabetes, obesity, diabetic complications or arteriosclerosis can do.
  • the expression of a gene is more enhanced or attenuated, it can be determined that type 2 diabetes, obesity, diabetic complications, or arteriosclerosis is more serious! In addition, if the gene expression is more enhanced or attenuated, it can be evaluated that the risk of developing type 2 diabetes, obesity, diabetic complications or arteriosclerosis is high.
  • gene expression products may be measured as well as directly measuring gene expression.
  • the gene expression product can be measured by using an antibody against the protein encoded by the gene.
  • the present invention also includes a protein encoded by a gene whose expression varies depending on the pathological condition between the liver of a type 2 diabetic or obese patient and the liver of a normal person, an antibody against the protein, a reagent containing these, and the like. To do.
  • the present invention provides detection (diagnosis) of type 2 diabetes, obesity, diabetic complications or arteriosclerosis for elucidation of the etiology of type 2 diabetes, obesity, diabetic complications or arteriosclerosis.
  • detection for human type 2 diabetes, obesity, diabetic complications or atherosclerosis, compared to liver tissue of normal humans, human type 2 diabetes, obesity, Increased or attenuated expression in liver tissue of patients with diabetic complications or arteriosclerosis
  • a reagent for measuring the expression of type 2 diabetes-related genes and Z or obesity-related genes which include nucleotides comprising a nucleotide sequence or a partial sequence thereof.
  • the reagent is a reagent comprising a nucleotide comprising the nucleotide sequence of the gene or a nucleotide comprising a partial sequence thereof as a probe or primer, and a nucleotide comprising the nucleotide sequence of the gene or a nucleotide comprising a partial sequence thereof is immobilized.
  • a phased substrate such as a microarray.
  • the microarray can be prepared by immobilizing a nucleotide having the nucleotide sequence of the gene or a nucleotide containing a partial sequence thereof on an appropriate substrate.
  • Examples of the fixed substrate include a glass plate, a quartz plate, and a silicon wafer.
  • a glass plate For example, 3.5mm x 5.5mm, 18mm x 18mm, 22mm x 75mm, etc. as the size of the board This is set according to the number of probe spots on the board and the size of the spots. can do.
  • a method for immobilizing a polynucleotide or a fragment thereof, using a nucleotide charge it can be electrostatically bound to a solid support surface-treated with a polycation such as polylysine, polyethyleneimine or polyalkylamine, Nucleotides introduced with functional groups such as amino group, aldehyde group, SH group, and pyotin can be covalently bonded to the solid phase surface introduced with functional groups such as amino group, aldehyde group, and epoxy group. Fixing may be performed using an array machine.
  • the reagent is at least one of 62 genes belonging to the HbAlc disease state-related gene group, HOMA-R disease state-related gene group, MCR disease state-related gene group and BMI disease state-related gene group, preferably at least two types, Preferably, it contains at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 30, more preferably all. In addition, at least one, preferably at least two of the genes belonging to the HbAlc disease-related gene group, HOMA-R disease-related gene group, MCR disease-related gene group, and BMI disease-related gene group, More preferably, it includes 5 types, more preferably at least 10 types, and even more preferably all.
  • these genes can serve as key molecules for pathological conditions such as glycolipid metabolism, diabetic complications, and arteriosclerosis. Therefore, the gene of the present invention and the reagent containing the gene are key molecules that produce pathologies such as sugar lipid metabolism, diabetic complications, and arteriosclerosis. Use as a tool for analysis, get.
  • detection of type 2 diabetes, obesity, diabetic complications or arteriosclerosis containing the above reagents, or risk of suffering from type 2 diabetes, obesity, diabetic complications or arteriosclerosis Detection agents for assessing are also encompassed by the present invention.
  • expression is increased or decreased in the liver tissue of patients with human type 2 diabetes, obesity, diabetic complications or arteriosclerosis, as compared to liver tissue of normal humans.
  • a drug that increases or decreases the expression of the gene can be selected as a therapeutic or prophylactic agent by administering the candidate drug to a subject or bringing the candidate drug into contact with the gene.
  • Data are mean ⁇ SD, AST, aspartate t raiisHJiiiB se; ALT, al anine auiinot rans feiase;
  • liver tissue was obtained from 21 patients with type 2 diabetes and 11 patients with normal glucose tolerance (Table 2). Liver tissue was obtained by percutaneous ultrasound-guided liver biopsy, and then immediately frozen in liquid nitrogen.
  • RNA extracted from liver tissue was adenylated using FastTrac mRNA extraction kit (Invitrogen, San Diego, CA). For each sample, 2.5 g of RNA was used in the SAGE method. The protocol for the S AGE method followed previous reports (Yamashita T. et al., Biochem Biophys Res Commun 269: 110-116, 2000; Yamashita T. et al., Biochem Biophys Res Comm un 282: 647-654, 2001). The SAGE library was sequenced using the ABI PRISM 377 DNA sequencer and the BigDye terminator single cycle sequencing kit (PE Biosystems, Foster city, CA). Sequenced files were analyzed using SAGE 2000 software (www.sagenet.org).
  • Double-stranded cDNA was used as a cage for quantitative real-time PCR.
  • ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif., USA) was used.
  • Primer and TaqMan probe set is from Applied Biosystems: C (Assays—on—Demand gene expression product).
  • the gene expression of the target sequence was standardized with the expression level of the endogenous control beta-actin RNA (TaqMan Control Reagent Kit, Appleid Biosystems). .
  • PCR conditions were 50 ° C for 2 minutes and 95 ° C for 10 minutes for 1 cycle, and then 95 ° C for 15 seconds and 60 ° C for 1 minute for 40 cycles.
  • HOMA-R is based on previous reports (Matthews DR et al., Diabetologia 28: 412-419, 1985). Fasting blood glucose level (mg / dL) X fasting blood insulin concentration in each case; z U / ml ) Calculated from / 405.
  • the unpaired-t test was used to compare the clinical characteristics of patients with type 2 diabetes and normal glucose tolerance.
  • the relationship between BMI, HOMA-R, MCR and the expression level of each gene was analyzed by linear correlation analysis. For V and deviation, P ⁇ 0.05 was considered significant.
  • the genes of 144,901 tags were identified.
  • Basic local alignment search tool program (BLAST (http://www.ncbi.nlm.nih.gov/BLAST/), and the corresponding gene was analyzed from the tag sequence. Discover and identify.
  • BLAST http://www.ncbi.nlm.nih.gov/BLAST/
  • the genes were classified according to the subcellular localization of transcripts. As a result, 232 genes were genes encoding "extracellular secretory proteins". It was.
  • genes that were upregulated 1.5 times or more in the type 2 diabetes patient group and 114 genes that were expressed 1.5 times or less in comparison with the group with normal glucose tolerance were identified.
  • the amount of gene expression in each case was measured using quantitative real-time PCR, and compared with clinical parameters such as BMI, H0MA-R, and MCR.
  • genes Six genes were identified that showed a significant correlation between expression levels and HOMA-R (Table 5). These genes vary in their expression in relation to insulin resistance in the liver. Among these genes are genes that regulate cell lipid metabolism and energy metabolism, or pathologies related to insulin resistance. It is expected to contain genes that form
  • expression is attenuated or enhanced in the liver of type 2 diabetic patients, and its expression level is glycemic control, obesity, liver 'skeleton Muscle insulin resistance
  • Correlated secretory protein coding genes were identified.
  • the liver-derived secretory protein encoded by these genes is thought to affect intracellular insulin signals, sugar and lipid metabolism, etc., and is related to systemic fat accumulation and the development of insulin hypotonia and arteriosclerosis. It is expected that elucidating the functions of these secreted proteins may lead to elucidation of the etiology of type 2 diabetes and obesity and the development of new therapies.

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Abstract

L’invention concerne un réactif qui permet de mesurer l’expression d’un gène dont les variations sont associées au diabète ou à l’obésité en utilisant un gène dont l’expression change en fonction des troubles du diabète, ainsi qu’un procédé permettant de détecter le diabète ou l’obésité en utilisant ce réactif. Selon cette invention, le réactif permet de mesurer l’expression d’un gène associé au diabète de type 2 et/ou d’un gène associé à l’obésité, qui contient des nucléotides composés d’une séquence de base d’au moins un gène sélectionné parmi le groupe comprenant des gènes dont l’expression augmente ou diminue dans un tissu hépatique d’un patient atteint d’un diabète humain de type 2 ou d’une obésité en comparaison d’un tissu hépatique d’un être humain normal et qui code pour une protéine sécrétoire ou des nucléotides contenant une séquence partielle correspondante.
PCT/JP2006/307700 2005-04-22 2006-04-12 Gene dont l’expression change en fonction des troubles du diabete et son utilisation Ceased WO2006115047A1 (fr)

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JP2005-125689 2005-04-22
JP2005125689A JP2006296346A (ja) 2005-04-22 2005-04-22 糖尿病の病態と関連して発現変動する遺伝子およびその利用

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CN101561440B (zh) * 2008-04-16 2014-03-05 中国科学院上海生命科学研究院 纤维胶凝蛋白3的应用
WO2011149057A1 (fr) * 2010-05-27 2011-12-01 国立大学法人 東京大学 Procédés de détection du risque d'obésité et du risque de déclenchement de diabète
JP7781057B2 (ja) * 2019-10-18 2025-12-05 リサーチ インスティチュート アット ネイションワイド チルドレンズ ホスピタル Ｉｒｆ２ｂｐｌ遺伝子の変異に関連する障害の治療のための材料および方法

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015025975A1 (fr) * 2013-08-23 2015-02-26 独立行政法人国立国際医療研究センター Méthode et trousse de détection d'un début ou d'un risque de début de néphropathie diabétique
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US11821905B2 (en) 2015-01-27 2023-11-21 Arterez, Inc. Biomarkers of vascular disease

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