HK1182769B

HK1182769B - Atopic dermatitis marker and technique of using the same

Info

Publication number: HK1182769B
Application number: HK13110154.6A
Authority: HK
Inventors: 宫田智; 宫崎香; 安田知永; 岩松明彦; 池泽善郎; 相原道子; 森山佳谷乃
Original assignee: 株式会社芳珂; 宫崎香; 池泽善郎; 相原道子; 森山佳谷乃
Priority date: 2005-10-21
Filing date: 2009-01-23
Publication date: 2015-10-23

Abstract

It is intended to search for a substance which is usable as a disease marker for atopic dermatitis. A method of evaluating atopic dermatitis which comprises measuring the expression of a specific protein and/or the expression of the gene thereof in skin cells and/or a skin tissue, wherein the specific protein shows a change in the expression thereof with the onset of atopic dermatitis or a change in the expression thereof depending on the degree of the causative factor for atopic dermatitis. It is also intended to provide a kit for evaluating the degree of inflammation in atopic dermatitis or the onset risk of atopic dermatitis and a method of identifying a substance which is efficacious in treating and/or preventing atopic dermatitis.

Description

Atopic dermatitis marker and technique of using same

The present application is a divisional application of the present application having an application number of 200680039191.6, application date of 2006, 10/19, and application names "atopic dermatitis markers and utilization techniques" of zeylake, kawasaki, kushishanlang, facia, and sanshan jianaian.

Technical Field

The present invention relates to an atopic dermatitis marker and a technique for using the same.

Background

Atopic dermatitis is a disease caused by various factors such as genetic factors and environmental factors, and its mechanism is not clear. Currently, atopic dermatitis is mainly diagnosed by visual observation, and is subjectively conscious in clinical medicine, and thus is lacking in objectivity. Particularly in atopic dermatitis, it is very difficult to determine the severity of rash that determines the treatment method. Further, since the society is increasingly keen on the trend of huge column steroid external preparations commonly used for atopic dermatitis treatment, it is desired to have an appropriate treatment method and an appropriate amount of the drug. Therefore, it is an important subject to search for an atopic dermatitis marker that can classify the severity of atopic dermatitis as an indication of the deterioration of the disease, an improvement index, or a therapeutic effect.

Currently, IgE is used as a marker for atopic dermatitis. However, since diseases showing an increase in the antibody titer of IgE include, in addition to atopic dermatitis, bronchial asthma, liver cirrhosis, and the like, the antibody value of IgE is not necessarily related to atopic dermatitis even if it is high. Recently, it has been pointed out that SCCA1 is a novel biomarker because of its high expression in the skin and blood of atopic dermatitis patients (non-patent document 1). There have been reports that NGF, Substance P (Substance P), IL-16 and the like are very effective as serum markers for atopic dermatitis patients (non-patent documents 2 and 3). In addition, there is a method of performing a comprehensive examination of biomarkers of atopic dermatitis using skin of inflammatory sites and non-inflammatory sites of atopic dermatitis. For example, a patent has been filed for identifying a disease marker of an atopic dermatitis-related gene based on gene expression analysis using a DNA microarray (patent documents 1 and 2). In addition, there is a report that an atopic dermatitis marker is identified by a mass spectrometer after culturing epidermal keratinocytes and fibroblasts obtained from skin tissues of atopic dermatitis patients, extracting proteins from the cells, separating the proteins by two-dimensional electrophoresis, and then analyzing the proteins (non-patent documents 4 and 5).

However, there has been no precedent to date for proteome analysis using proteins in the skin tissue of atopic dermatitis model mice. In addition, since proteome analysis of human atopic dermatitis is performed by collecting cells from the skin and culturing the cells in the primary culture, it is hard to say that the state of human atopic dermatitis is reflected immediately.

Patent document 1 Japanese laid-open patent publication No. 2005-110602

Patent document 2 International publication No. WO 01-065259 pampthet

Non-patent document 1: Clin. exp. Allergy2005;35:1327-

Non-patent document 2 Br.J.Dermatol.2002Jul;147(1):71-79

Non-patent document 3 Br.J.Dermatol.2006Jun;154(6):1112-1117

Non-patent document 4: Proteomics2004, 4, 3446-

Non-patent document 5 Proteomi cs2006, 6, 1362-

Disclosure of Invention

The purpose of the present invention is to provide a substance that is effective as an objective marker for atopic dermatitis in diagnosis and treatment. And an object of the present invention is to provide a method for screening a component effective for the prevention or treatment of atopic dermatitis using an atopic dermatitis marker.

The present inventors applied hapten to an atopic dermatitis model mouse (NC/Nga mouse) to artificially induce atopic dermatitis. Lactoferrin treatment, which is known to inhibit atopic dermatitis, is also performed to inhibit inflammation. The skin tissue at this time was used to measure proteins that change due to the onset of atopic dermatitis. The analysis method was proteome analysis using two-dimensional electrophoresis (2-DE) and Mass Spectrometry (MS), and the identified proteins were further confirmed for changes by immunoblotting.

As a result, FABP-5, Apolipoprotein A1(Apolipoprotein A1), Vimentin (Vimentin), Rho GDI, and the like were identified as proteins whose expression was increased in association with the onset of atopic dermatitis. In addition, as proteins whose expression is reduced in association with inflammation of atopic dermatitis, Galectin (Galectin) such as Galectin-1, -3, -4, -7, -8(Galectin-1, -3, -4, -7, -8), Desmin (Desmin) which is a cytoskeletal protein, Moesin (Moesin), Ezrin (Ezrin), Radixin (radiaxin), Annexin II (Annexin II), Enolase 1(Enolase1), FABP-4, PARK7, HSP70, HSP90 and the like have been identified. Most of these proteins showed opposite changes when they were treated with lactoferrin after hapten treatment, indicating that the presence or absence of atopic dermatitis had a good correlation with the increase or decrease in expression.

In order to analyze the correlation between the atopic dermatitis markers and the degree of inflammation of human atopic dermatitis, as described above, the horny layer was collected from atopic dermatitis patients and healthy volunteers using a horny layer tester (checker) capable of collecting the horny layer noninvasively, safely and easily, proteins were extracted therefrom, and the expression of the atopic dermatitis markers was measured by Western blotting. As a result, it was confirmed that the expression of serum albumin (Serumalbumin), immunoglobulin G (immunoglobulin G), Annexin II (Annexin II), Apolipoprotein A1(Apolipoprotein A1), FABP-5, Enolase 1(Enolase1) and Galectin-7 (Galectin-7) was also increased in association with the increase in the degree of inflammation of atopic dermatitis. In addition, arginase i (arginase i) and Uracil DNA glycosylase (Uracil-DNA glycosylase) were identified as proteins whose expression was decreased with an increase in the degree of inflammation of atopic dermatitis.

The main contents of the present invention are as follows.

1. A method for determining atopic dermatitis, which comprises measuring the expression of a specific protein and/or the gene expression thereof in skin cells and/or skin tissues, wherein the expression of the specific protein is changed with inflammation of atopic dermatitis.

The specific proteins are 23 of the following types:

(1) a protein selected from the Galectin group consisting of Galectin-1 (Galectin-1), Galectin-3 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7) and Galectin-8 (Galectin-8),

(2) proteins selected from the HSP group consisting of HSP70 and HSP90,

(3) a protein selected from the group consisting of Vimentin (Vimentin), Rho GDI, Desmin (Desmin), Moesin (Moesin), Ezrin (Ezrin), and Radixin (radiaxin),

(4) a protein selected from the FABP group consisting of FABP-4 and FABP-5,

(5) in addition, Serum albumin (Serum albumin), immunoglobulin G (immunoglobulin G), Annexin II (Annexin II), Apolipoprotein A1(Apolipoprotein A1), Enolase 1(Enolase1), PARK7, arginase I (ArginaseI), and Uracil DNA glycosylase (Uracil DNA glycosylase).

2.1. The method of (1), wherein the skin cells and/or skin tissues are stratum corneum of the skin collected with a keratometer.

3.1. The method of (1), which determines the expression of a specific protein at the protein level.

4.1. The method of (1), which measures the gene expression of a specific protein at the RNA level.

5. A kit for determining atopic dermatitis, which comprises a reagent for measuring the expression of a specific protein in skin cells and/or skin tissues, the expression of which changes with inflammation in atopic dermatitis.

The specific proteins are 23 of the following types:

(2) proteins selected from the HSP group consisting of HSP70 and HSP90,

(4) a protein selected from the FABP group consisting of FABP-4 and FABP-5,

(5) in addition, Serum albumin (Serum albumin), immunoglobulin G (immunoglobulin G), Annexin II (Annexin II), Apolipoprotein A1(Apolipoprotein A1), Enolase 1(Enolase1), PARK7, arginase I (arginase I), and Uracil DNA glycosylase (Uracil DNA glycosylase).

6.5. The kit of (1), which is provided with a keratometer for collecting skin cells and/or skin tissues.

7.5. The kit as described in (1), wherein the reagent is an antibody specifically recognizing a specific protein whose expression is changed accompanying inflammation of atopic dermatitis.

8.5. The kit as described in (1), wherein the reagent is a nucleic acid probe capable of specifically hybridizing to mRNA of a specific protein whose expression is changed in association with inflammation of atopic dermatitis.

9.5. The kit as described in (1), wherein the reagent is1 pair of nucleic acid primers consisting of a nucleic acid primer capable of specifically hybridizing to mRNA of a specific protein whose expression is changed in association with inflammation of atopic dermatitis and a nucleic acid primer capable of specifically hybridizing to cDNA synthesized using the mRNA as a template.

10. A method for identifying a substance having an effect on the treatment and/or prevention of atopic dermatitis, comprising the steps of:

(a) bringing skin cells and/or skin tissue into contact with a substance to be measured,

(b) culturing the skin cells and/or skin tissues contacted with the analyte in step (a) for a predetermined period of time,

(c) measuring the expression of a specific protein and/or the gene expression thereof in the skin cells and/or skin tissues cultured in the step (b), wherein the expression of the specific protein is changed with the inflammation of atopic dermatitis, and

(d) and (c) comparing the expression of the specific protein and/or the gene expression thereof measured in the step (c) with the expression of the specific protein and/or the gene expression thereof in the skin cell and/or the skin tissue as a control, and evaluating the effect of the test substance on the expression of the specific protein and/or the gene expression thereof in the skin cell and/or the skin tissue.

The specific proteins are 23 of the following types:

(2) proteins selected from the HSP group consisting of HSP70 and HSP90,

(4) a protein selected from the FABP group consisting of FABP-4 and FABP-5,

The present invention makes it possible to establish an evaluation system for diagnosing atopic dermatitis and measuring the severity thereof. By using this evaluation system, the severity and the risk of atopic dermatitis can be determined more objectively than by conventional methods. In addition, the present invention provides a kit for determining the severity and risk of atopic dermatitis and a method for identifying a component having an effect of inhibiting inflammation of atopic dermatitis.

Drawings

Fig. 1 shows the state of mouse skin using 4 experimental conditions ((1) hapten (-)/lactoferrin (-), (2) hapten (-)/lactoferrin (+), (3) hapten (+)/lactoferrin (-), (4) hapten (+)/lactoferrin (+)).

Fig. 2(a) shows the results of two-dimensional electrophoresis using skin tissues of atopic dermatitis model mice (experimental conditions for hapten (-)/lactoferrin (-) and hapten (+)/lactoferrin (-).

Fig. 2(b) shows the results of two-dimensional electrophoresis using skin tissues of atopic dermatitis model mice (experimental conditions for hapten (-)/lactoferrin (+) and hapten (+)/lactoferrin (+).

FIG. 3 shows the change in expression of galectin in skin tissues of atopic dermatitis model mice.

FIG. 4 shows changes in the expression of cytoskeletal proteins and HSP proteins in skin tissues of mice model atopic dermatitis.

FIG. 5 shows changes in the expression of proteins in skin tissues of mice model atopic dermatitis.

FIG. 6 shows the results of identification of proteins whose expression was increased in a sample with onset of atopic dermatitis using a mass spectrometer (MALDI TOF-MS).

FIG. 7 shows changes in the expression of proteins in skin tissues of atopic dermatitis patients.

FIG. 8(a) and (b) shows the correlation between the severity of atopic dermatitis patients and the protein expression level.

FIG. 9(a) and (b) show a summary of the results of quantifying the expression intensities of 5 marker proteins, namely, Enolase 1(Enolase1), FABP-5, SCCA2, Apolipoprotein A1(Apolipoprotein A1) and serum albumin (Serumalbumin), in each sample, depending on the severity of the patient.

FIGS. 10(a) - (f) show the correlation between the amount of transdermal water loss and the amount of expression of the marker in healthy subjects and humans with a history of atopic dermatitis.

FIGS. 11(a) - (f) show the correlation between keratinocyte area and marker expression levels in healthy subjects and in persons with a history of atopic dermatitis.

Detailed Description

The embodiments of the present invention will be described in more detail below.

The present invention provides a method for determining atopic dermatitis, which comprises measuring the expression of a specific protein and/or the gene expression thereof in skin cells and/or skin tissues. The expression of specific proteins is changed accompanying inflammation of atopic dermatitis.

The expression may be modified by the presence or absence of expression of the protein and/or its gene, or by the increase or decrease of the expression level.

The specific protein is preferably selected from the group consisting of Apolipoprotein A1(Apolipoprotein A1), FABP-4, FABP-5, Vimentin (Vimentin), Rho GDI, Annexin II (Annexin II), Enolase 1(Enolase1), Galectin-1 (Galectin-1), Galectin-3 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7), Galectin-8 (Galectin-8), PARK7, Desmin (Desmin), Moesin (Moesin), Ezrin (Ezrin), rootprotein (Radixin), HSP70, HSP90, Serum albumin (Serum albumin), immunoglobulin G (immunoglobulin G (Immunoglobulin G), arginase I (Arginesei), and Uracil glycosylase (Uracil-glycosylDNA). The selected protein may be 1 kind or plural kinds.

The 23 specific proteins are shown below:

(1) a protein selected from the group consisting of Galectin (Galectin) consisting of Galectin-1 (Galectin-1), Galectin-3 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7) and Galectin-8 (Galectin-8),

(2) proteins selected from the HSP group consisting of HSP70 and HSP90,

(4) a protein selected from the FABP group consisting of FABP-4 and FABP-5,

Apolipoprotein a1(Apolipoprotein a1) is a secreted protein with a molecular mass of 30,778Da and acts to promote the efflux of cholesterol from tissues or the reverse transport of cholesterol to the liver. It is known as a secreted protein present in large amounts in chylomicrons and plasma high density lipoprotein (plasma HDL), and is expressed in psoriasis (characterized by the regular thickening of the epidermis and parakeratosis) in skin tissues. Gene sequence information (Apolipoprotein A1, Nucleic Acids Res.11:2827-2837(1983), P02647). Amino acid sequence information (Apolipoprotein A1, biochem. Biophys. Res. Commun.80:623-630(1978), M27875).

FABP-5 (Fatty acid binding protein-5: Fatty binding protein-5) is an intracellular protein with a molecular mass of 15,033Da, is a protein mainly present in the epidermis, and is identified by increased expression in tissues of psoriasis (characterized by regular hypertrophy and parakeratosis of the epidermis). Is an intracellular protein that binds to fatty acids (oleic acid) and hydrophobic ligands, and is involved in the uptake, transport, and metabolism of fatty acids, as well as in the proliferation and differentiation of cells. The expression of FABP-5 by epidermal keratinocytes during proliferation was low, but increased by about 2-fold through differentiation induction. Binds to S100a7 (calcium-dependent signaling protein) highly expressed in psoriasis, and localizes in focal adhesion (focaldhese) like structures in the differentiation stage of epidermal keratinocytes. Gene sequence information (Fatty acid binding protein 5, J.invest.Dermatol.99:299-305(1992), BC 070303). Amino acid sequence information (Fatty acid-binding protein, epidermal, biochem. J.302:363-371(1994), Q01469).

Vimentin (Vimentin) is a cytoskeletal protein with a molecular mass of 53,520Da, and is a protein constituting classIII type intermediate filament. And the cell is distributed in a wide range of net shape in a plurality of mesenchymal cells, and the cell is endowed with strength to respond to external mechanical pressure. Its structure is regulated by phosphorylation. Binding proteins of reticulin, and catenin (synemin) are also known to exist. Gene sequence information (Vimentin, Nucleic Acids Res.18:6692-6692(1990), M14144). Amino acid sequence information (Vimentin, Electrophoresis18:588-598(1997), P08670).

Rho GDI (RhoGDP dissociation inhibitor1) (RhoGDP-dissociation inhibitor1) is an intracellular protein with the molecular mass of 23,207Da, is highly expressed when epidermal keratinocytes are differentiated, and can inhibitively regulate the conversion of Rho protein from GDP to GTP. Therefore, Rho GDI is forcibly expressed in cells to eliminate stress fibers, adhesion spots, etc., thereby disrupting the intracellular actin skeleton system. Gene sequence information (RhoGDP-dissociationInhibitor1, exp. cell Res.209:165-174(1993), X69550). Amino acid sequence information (RhoGDP-association inhibitor, P52565).

Annexin II (Annexin II) is an intracellular protein with a molecular mass of 38,473Da, and a part of it is reported to be secreted extracellularly. It is a calcium-controlled membrane-bound protein that binds two calcium ions, localized near the cell membrane. 1 of the 2 groups of annexin (annexin) repeats binds calcium and 1 binds phospholipid. This protein is either cross-linked with actin, cytoskeletal proteins bound to phospholipids on the cell membrane, or activates plasminogen by tPA (tissue plasminogen activator). Gene base sequence information (Annexin A2, Gene95:243-251(1990), BC 015834). Amino acid sequence information (Annexin A2, J.biol.chem.266:5169-5176(1991), P07355).

Enolase 1(Enolase1) is an intracellular protein with a molecular mass of 47,038Da, with 2-phospho-D-glycerate = phosphoenolpyruvate + H₂O(2-phospho-D-glycerate=phosphoenolpyruvate+H₂O) in a glycolytic system. Some of them have been reported to be associated with cell proliferation and allergy. In leukocytes and nerves, activation of plasminogen on the cell surface is controlled, and is involved in the formation of fibrin. Magnesium is required to have a stable dimer structure. Although localized within the cytoplasm, only homodimers were present in the cell membrane. α -enolase (α -enolase) is expressed in most tissues, β -enolase (β -enolase) is expressed only in muscle tissue, and γ -enolase (γ -enolase) is expressed only in nerve tissue. Gene sequence information (Alpha enolase, Proc. Natl. Acad. Sci. USA.83:6741-6745(1986), M14328). Amino acid sequence information (Alpha enolase, Enzyme Protein48:37-44 (C.))1995)，P06733)。

Galectin-1 (Galectin-1) is an intracellular protein having a molecular mass of 14,585Da, is also secreted extracellularly, and is present in the heart, stomach, skeletal muscle, nerve, thymus, kidney, placenta, and the like. Binding to beta-galactoside, CD45, CD3, C D4 and the like. It is known to affect cell proliferation promoting action, induction of cell death and immune response. In addition, specific binding to components of extracellular matrix such as laminin and integrin, or cell receptors, greatly affects cell adhesion and migration. Gene sequence information (Galectin-1, J.biol.chem.264:1310-1316(1989), BT 006775). Amino acid sequence information (Galectin-1, J.biochem.104:1-4(1988), P09382).

Galectin-3 (Galectin-3) is an intracellular protein with molecular mass of 35,678Da, is a galactose-specific lectin bound to IgE, and is mainly expressed in the epithelium of the large intestine and activated macrophages. Galectin-3 (Galectin-3) is reported to be produced by epidermal keratinocytes, present on the surface of islet cells of the skin, bind to IgE and regulate the immune system. Gene sequence information (Galectin-3, Proc. Natl. Acad. Sci. USA.87:7324-7328(1990), AB 006780). Amino acid sequence information (Galectin-3, P17931).

Galectin-4 (Galectin-4) is an intracellular protein with a molecular mass of 35,941 Da. Galectin-4 (Galectin-4) was isolated from the parahaemocarcinoma cell line T84 and was present at the site of cell-to-stroma adhesion and at the site of intercellular adhesion. Gene sequence information (Galectin-4, Eur.J.biochem.248:225-230(1997), AB 006781). Amino acid sequence information (Galectin-4, P56470).

Galectin-7 (Galectin-7) is an intracellular protein with a molecular mass of 14,944 Da. In general, it is a protein involved in apoptosis that controls cell proliferation between cells, between cells and extracellular matrix, and controls JNK activity and cytochrome C release. It is also secreted to cytoplasm, nucleus and extracellular space. Is a member of the Galectin subfamily that was first cloned in human epidermis, and it was found from studies on cultured epidermal keratinocytes that Galectin 7(Galectin-7) is expressed in all epidermal cells without being affected by the degree of keratinization. Gene sequence information (Galectin-7, Dev. biol.168:259-271(1995), L07769). Amino acid sequence information (Galectin-7, J.biol.chem.270:5823-5829, 1995, P47929).

Galectin-8 (Galectin-8) is an intracellular protein with a molecular mass of 35,539 Da. Galectin-8 (Galectin-8) is expressed in many tissues such as liver, heart, muscle, kidney, brain, etc., and is specifically expressed in prostate cancer, but is not present in normal prostate and benign prostatic hypertrophy. Essentially absent from the embryo, but very high expression in mature individual tissues. Gene sequence information (Galectin-8, Proc. Natl. Acad. Sci. U.S.A.93:7252-7257(1996), X91790). Amino acid sequence information (Galectin-8, O00214).

PARK7 is an intracellular protein with a molecular mass of 19,891 Da. PARK7 was identified as a protein associated with Parkinson's disease, but it was also subsequently shown to be involved in re-epithelialization (re-epithelialization) in the skin wound healing process. And also suggests that it may function as a protease in view of its steric structure. Gene sequence information (Parkinsondisease7, biochem. Biophys. Res. Commun.231:509-513(1997), BC 008188). Amino acid sequence information (DJ-1protein, Q99497).

Desmin (Desmin) is a cytoskeletal protein with molecular mass of 53,405Da, a class iii intermediate filament that plays a role in maintaining cell structure and strength. Is a polymer of fibrous polypeptide, and is present in smooth muscle and striated muscle. Gene sequence information (Desmin, Gene78:243-254(1989), U59167). Amino acid sequence information (Desmin, P17661).

Ezrin (Ezrin) is an intracellular protein with a molecular mass of 69,268 Da. The Moesin and the radxin (radiaxin) proteins are homologous molecules. Mainly plays a role in connecting cytoskeletal proteins and cell membranes. Located inside the filamentous processes called microvilli (microvilli) of the cell membrane, the microvilli (microvilli) constituting the intestinal epithelial cells are phosphorylated by tyrosine kinases. Gene sequence information (Ezrin, J.biol.chem.264: 16727-. Amino acid sequence information (Ezrin, biochem. Biophys. Res. Commun.224:666-674(1996), P15311).

Root protein (radiaxin) is an intracellular protein with a molecular mass of 68,564 Da. Mainly plays a role in connecting cytoskeletal proteins and cell membranes. Gene sequence information (Radixin, Genomics16:199-206(1993), L02320). Amino acid sequence information (radiaxin, P35241).

Moesin (Moesin) is an intracellular protein with a molecular mass of 67,689 Da. Mainly plays a role in connecting cytoskeletal proteins and cell membranes. Reduced expression was reported in abnormally differentiated epidermal keratinocytes. Gene sequence information (Moesin, Proc. Natl. Acad. Sci. USA.88:8297-8301(1991), M69066). Amino acid sequence information (Moesin, Proc. Natl. Acad. Sci. USA.88:8297-8301(1991), P26038).

FABP-4 (Fatty acid binding protein-4: fat acid-binding protein-4) is an intracellular protein with a molecular mass of 14,588 Da. Has the function of delivering lipid to fat cells, mainly combines with fatty acid and retinoic acid in cells, increases the expression of differentiation stages depending on fat cells, and has high homology with FABP-5. Gene sequence information (Fatty acid-binding protein, adipocyte, Biochemistry28:8683-8690(1989), BT 006809). Amino acid sequence information (Fatty acid-binding protein, adipocyte, P15090).

HSP70 (Heat shock protein 70: Heat shock protein70) is an intracellular protein with a molecular mass of 70,052Da, and generally functions as a molecular chaperone in cells and participates in folding, transportation, aggregation, degradation and the like of proteins. Moreover, the role of HSP70 in mitochondria and ER is to make protein normally transfer, and the HSP70 is also cooperated with HSP90 to participate in signal transmission in cells. Information on the base sequence of the gene (Heat shock70kDa protein1A, Immunogenetics32:242-251(1990), BC 002453). Amino acid sequence information (Heat shock70kDa protein1, P08107).

HSP90 (Heat shock protein: Heat shock protein90) is an intracellular protein with a molecular mass of 85,453 Da. HSP90 changes structure when cells are subjected to high temperatures, acting as a chaperone to prevent irreversible denaturation of other proteins. It is also reported that signal transduction is involved in certain cells. Gene base sequence information (Heat shock protein90, Nucleic acids sRs.17: 7108-7108(1989), X15183) amino acid sequence information (Heat shock protein90, J.biol.chem.264:2431-2437(1989), P07900).

Serum albumin (Serum albumin) is a secreted protein with a molecular mass of 69,367Da that is contained in large amounts in Serum. The essential functions of Serum albumin (Serum albumin) are the maintenance of the oncotic pressure of the blood and the transport of various substances. Gene sequence information (Serum albumin, Proc. Natl. Acad. Sci. U.S.A.79:71-75(1982), V00494). Amino acid sequence information (Serum albumin, FEBS Lett.58:134-137(1975), P02768).

IgG, immunoglobulin G (immunoglobulin G) is a secreted protein with the molecular mass of 42,632Da and accounts for 75 percent of the total human immunoglobulin. The physiological activities of IgG include complement activation, macrophage uptake enhancement, placenta permeability, and the like. Immunoglobulin g (immunoglobulin g) is a large subunit (H chain) of immunoglobulin IgG and is expressed by gene class conversion. It is abundant in serum, has a long half-life, forms various antibody molecules together with a light chain (L chain), and plays a central role in immune response. Gene sequence information (Immunoglobulin gamma, Nucleic acids SRes.16:11824-11824(1988), X14356). Amino acid sequence information (Immoglobulin gamma, Protein Sci.13:2819-2824 (2004.), P12314).

Arginase I (ArginaseI) is an intracellular protein with a molecular mass of 34,735Da and is a one-way reaction enzyme that hydrolyzes L-arginine into L-ornithine and urea. It is localized in liver, and exists in kidney, brain, mammary gland and skin in trace amount. The deficiency may cause argininemia, mental retardation, and spastic quadriplegia. Gene sequence information (Arginase type I, Proc. Natl. Acad. Sci. U.S.A.84: 412. 415(1987), AY 074488). Amino acid sequence information (Arginase-1, P05089).

Uracil DNA glycosylase (Uracil-DNA glycosylase) is an intracellular protein with a molecular mass of 34,645 Da. When cytosine in DNA is deaminated to form uracil, it forms base pairs with adenine to induce mutations. To prevent this, uracil DNA glycosylase hydrolyzes the N-glycosidic bond of deoxyuridine formed in deamination. There are 2 subtypes. Subtype I is localized intracellularly to the mitochondria and as tissue distribution in the muscle. Subtype II is abundantly expressed in proliferating tissue nuclei. Gene sequence information (Uracil-DNA glycosylase, EMBO J.8:3121-3125(1989), X15653). Amino acid sequence information (Uracil-DNAycosylase, EMBO J.8:3121-3125(1989), P13051).

The protein may be a precursor protein, a mature protein, a cleaved protein or an uncleaved protein. The precursor protein may be a preprotein, preproprotein. Some of the preproteins and preproproteins also have a signal peptide.

The method of the present invention may measure the expression of a specific protein in skin cells and/or skin tissues, or may measure the gene expression thereof. For example, it can be determined by Northern blotting, RT-PCR, Western blotting, immunohistochemical analysis, ELISA, antibody chip, cDNA microarray, Fluorescence Resonance Energy Transfer (FRET) assay, etc.

In order to measure the expression of a specific protein at the protein level, an antibody that specifically recognizes the protein to be measured may be used. The antibody may be either a monoclonal antibody or a polyclonal antibody. These antibodies can be produced by a known method or can be commercially available. Antibodies can be detected by Western blotting¹²⁵I protein marker A, peroxidase-bound IgG, etc. are subjected to secondary detection. Antibodies may be labeled with fluorescent pigments, ferritin, enzymes, and the like, as determined by immunohistochemical analysis.

For measuring the gene expression of a specific protein at the RNA level, a nucleic acid probe capable of specifically hybridizing to mRNA of the protein to be measured (when measured by Northern blotting) or a pair of nucleic acid primers capable of specifically hybridizing to mRNA of the protein to be measured and a cDNA synthesized using the mRNA as a template (when measured by PCR) may be used. The nucleic acid probe and the nucleic acid primer can be designed based on the genetic information of the protein to be measured. The nucleic acid probe is preferably about 15 to 1500 bases. The nucleic acid probe may be labeled with a radioactive element, a fluorescent dye, an enzyme, or the like. The nucleic acid primer is preferably about 15 to 30 bases.

In the present invention, the presence or absence of expression of a specific protein and/or gene expression thereof in skin cells and/or skin tissues may be detected, or the expression level may be measured. The presence or absence of the expression of the protein and/or its mRNA can be confirmed by the presence or absence of spots or bands at predetermined positions. The expression level of the protein and/or its mRNA can be measured by the intensity of staining spots or bands. Alternatively, the protein and/or its mRNA may be quantified. For simultaneous detection of expression of a plurality of genes and expression of a plurality of proteins, a DNA array (probes immobilized on a substrate) (NATURE REVIEWS, DRUG DISCOVERY, VOLUME1, DECEMBER2002, 951-.

The skin cell and the skin tissue may be derived from a subject to be determined, and the biological species of the subject may be a mammal such as a human, a pig, a monkey, a gorilla, a dog, a cow, a rabbit, a rat, or a mouse.

By the method of the present invention, in order to determine atopic dermatitis, a skin biopsy sample or skin culture cells, skin culture tissues, or the like obtained therefrom may be used. The skin biopsy sample can be obtained by using a sample obtained by picking the stratum corneum layer of the skin with an adhesive tape (keratometer), as described in examples described later.

Examples of skin cells include epidermal keratinocytes, dermal fibroblasts, islet cells, melanocytes, adipocytes, endothelial cells, sebocytes, dermal papilla cells, and hair mother cells. Skin cells can be collected from the skin by a known method (novel cell culture experiment for molecular biology research, p57-71, sheep soil society, 1999) [ native name of Japanese: "novel cell culture experiment for molecular biology research ため", p57-71, sheep soil society, 1999 ] ].

Examples of the skin tissue include the stratum corneum, epidermis, and dermis of the skin. Skin tissue can be harvested from skin by well-known methods (acta. derm. venereol., 85, 389-.

The skin biopsy sample may be a cell or a tissue. Skin biopsy samples, such as stratum corneum collected using an adhesive tape of a keratometer or the like, are described in examples below. A keratology tester has been used for sampling a stratum corneum sample for measuring keratosis incompetence and cell area of the stratum corneum, and evaluating roughness of the skin and renewal rate of the stratum corneum [ (advancement of cosmetic usefulness and evaluation technology and future prospect, japanese cosmetic technologist society, pharmaceutical japanese newspaper, p95-96) [ japanese original name: "usefulness of cosmetics value technology and future prospect of と, japanese cosmetic society, japanese medicine , p95-96) ]. The method is very effective as a method for obtaining proteins noninvasively from the horny layer by collecting a sample of the horny layer easily and safely at home in a counseling (counseling) shop.

As one example of the present invention, atopic dermatitis can be judged by the following criteria.

As shown in the analysis examples using various antibodies such as Enolase 1(Enolase1), FABP-5, SCCA2, Apolipoprotein a1(Apolipoprotein a1), albumin (albumin), Annexin II (Annexin II) and the like shown in example 1 (fig. 8), skin samples were obtained from volunteers with atopic dermatitis onset, and the expression levels of various marker proteins or the expression levels of their genes were measured to reveal the relationship between the pathological condition and the expression level.

The degree of atopic dermatitis symptoms and the cause of the onset of atopic dermatitis in the test sample are determined by comparing the expression levels of the affected part (test sample) of the atopic dermatitis-affected person with the non-affected part (non-affected person control sample) and the same part (non-affected person control sample) of the atopic dermatitis-non-affected person. Thus, the analysis result of the marker protein can contribute to the diagnosis and treatment of atopic dermatitis exhibiting various symptoms.

The present invention also provides a kit for determining atopic dermatitis. The kit of the present invention comprises a reagent for measuring the expression of a specific protein in skin cells and/or skin tissues, the expression of which changes with inflammation caused by atopic dermatitis.

For example, the kit of the present invention contains a reagent for an antibody that specifically recognizes a specific protein whose expression is changed in association with inflammation of atopic dermatitis.

The specific protein is preferably selected from the group consisting of Apolipoprotein A1(Apolipoprotein A1), FABP-4, FABP-5, Vimentin (Vimentin), Rho GDI, Annexin II (Annexin II), Enolase 1(Enolase1), Galectin-1 (Galectin-1), Galectin-3 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7), Galectin-8 (Galectin-8), PARK7, Desmin (Desmin), Moesin (Moesin), Ezrin (Ezrin), rootprotein (Radixin), HSP70, HSP90, Serum albumin (Serum albumin), immunoglobulin G (immunoglobulin G (Immunoglobulin G), arginase I (Arginesei), and Uracil glycosylase (Uracil glycosylDNA).

The kit can also comprise adhesive tapes for collecting skin tissues, complete sets of reagents for detecting proteins on the adhesive tapes by an immunochemical method, instructions for use and the like. The instruction for use may include a method of using the kit, and a standard for determination of atopic dermatitis.

In another example, the kit of the present invention includes, as a reagent, a nucleic acid probe capable of specifically hybridizing to mRNA of a specific protein whose expression is changed in association with inflammation of atopic dermatitis.

The specific protein is preferably selected from the group consisting of Apolipoprotein A1(Apolipoprotein A1), FABP-4, FABP-5, Vimentin (Vimentin), Rho GDI, Annexin II (Annexin II), Enolase 1(Enolase1), Galectin-1 (Galectin-1), Galectin-3 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7), Galectin-8 (Galectin-8), PARK7, Desmin (Desmin), Moesin (Moesin), Ezrin (Ezrin), rootprotein (Radixin), HSP70, HSP90, Serum albumin (Serum albumin), immunoglobulin G (immunoglobulin G (Immunoglobulin G), arginase I (ArginaseI), and uracil glycosylase (uracilglycosylDNA).

The kit may further comprise an adhesive tape for collecting skin tissue, reagents for extracting RNA from skin tissue on the adhesive tape, reagents for analyzing RNA by Northern blotting, instructions for use, and the like. In the instruction manual, a method of using the kit is described, and a standard for determination of atopic dermatitis and the like are also described.

In another example, the kit of the present invention comprises a pair of nucleic acid primers consisting of a nucleic acid primer capable of specifically hybridizing to an mRNA of a specific protein whose expression is changed in association with inflammation of atopic dermatitis as a reagent and a nucleic acid primer capable of specifically hybridizing to a cDNA synthesized using the mRNA as a template.

The specific protein is preferably selected from the group consisting of Apolipoprotein A1(Apolipoprotein A1), FABP-4, FABP-5, Vimentin (Vimentin), Rho GDI, Annexin II (Annexin II), Enolase 1(Enolase1), Galectin-1 (Galectin-1), Galectin-3 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7), Galectin-8 (Galectin-8), PARK7, Desmin (Desmin), Moesin (Moesin), Ezrin (Ezrin), radicin (Radixin), HSP70, HSP90, Serum albumin (Serum albumin album), immunoglobulin G (immunoglobulin G Immunoglobulin G), arginase I (ArginaseI), and Uracil DNA glycosylase (Uracil).

The kit may further comprise an adhesive tape for collecting skin tissue, reagents for extracting RNA from skin tissue on the adhesive tape, reagents for analyzing RNA by RT-PCR, instructions for use, and the like. In the instruction manual, a method of using the kit is described, and a standard for determination of atopic dermatitis and the like are also described.

The present invention also provides a method for identifying a substance having an effect of treating and/or preventing atopic dermatitis, the method comprising the steps of:

The substance to be tested may be any substance, and examples thereof include proteins, peptides, vitamins, hormones, polysaccharides, oligosaccharides, monosaccharides, low-molecular compounds, nucleic acids (DNA, RNA, oligonucleotides, mononucleotides, etc.), lipids, natural compounds other than the above, synthetic compounds, plant extracts, isolates of plant extracts, and mixtures thereof.

Skin cells and skin tissues are as described above.

The contact between the test substance and the skin cells and/or the skin tissue may be carried out by any method, for example, a method of adding the test substance to a culture solution of the skin cells and/or the skin tissue, a method of culturing the skin cells and/or the skin tissue on a culture vessel or a culture plate (sheet) on which the test substance is applied or immobilized, or the like. The method may be a method of directly applying the test substance to the skin or a method of orally administering the test substance to the skin using a living body such as a human or a mammal other than a human (e.g., a mouse, a rat, a guinea pig, a rabbit, a pig, etc.).

The culture time of the skin cells and/or skin tissue is not particularly limited, and may be any time that can confirm the presence or absence of the effect of the test substance on the expression of the specific protein and/or the gene expression in the skin cells and/or skin tissue.

For example, when human normal epidermal keratinocytes are used as skin cells, the time is preferably 12 to 48 hours, more preferably 12 to 24 hours. Here, "culturing skin cells and/or skin tissue" means growing, developing and proliferating skin cells and/or skin tissue, and includes, in addition to growing, developing and proliferating skin cells and/or skin tissue after isolation, living, feeding, and culturing living bodies having skin cells and skin tissue, and the like.

The skin cells and/or skin tissue of the comparative control may be skin cells and/or skin tissue before contact with the test substance, or skin cells and/or skin tissue treated in the same manner except that they are not in contact with the test substance.

In one example of the present invention, when the expression level of any one of Apolipoprotein a1(Apolipoprotein a1), FABP-5, Vimentin (Vimentin), Rho GDI, Annexin II (Annexin II), Enolase 1(Enolase1), serum albumin (serum albumin) or immunoglobulin g (immunoglobulin g) in skin cells and/or skin tissues which have been brought into contact with a test substance is decreased as compared to a control, and the test substance is evaluated as having an effect of reducing the expression level of any one of the above proteins, the test substance can be identified as a substance having an effect of treating and/or preventing atopic dermatitis.

In another embodiment of the present invention, in skin cells and/or skin tissues that have been brought into contact with a test substance, the expression amount of FABP-4, Galectin-1 (Galectin-1), Galectin-3 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7), Galectin-8 (Galectin-8), PARK7, Desmin (Desmin), Moesin (Moesin), Ezrin (Ezrin), radxin (Radixin), HSP70, HSP90, arginase I (ArginaseI) or Uracil DNA glycosylase (Uracil DNA glycosylase) was increased as compared with the control, when the test substance is evaluated to have an effect of increasing the expression of any of the above proteins, the test substance can be identified as a substance having an effect of treating and/or preventing atopic dermatitis.

Examples

The present invention will be described in detail below with reference to examples, but the present invention is not limited to the examples.

Example 1

Materials and Experimental methods

1. Hapten treatment of atopic dermatitis model mice (NC/Nga mice)

An atopic dermatitis model mouse (NC/Nga mouse), a male mouse [ NC/Ngaslc, Sankyo Labo Service Corporation (japanese original name "kyowata ラボサービス co.") 6 weeks old was purchased and bred under conventional conditions. NC/Nga mice have a number of systems, and the system used in this study is a system that causes atopic dermatitis not to develop only when raised under ordinary circumstances, but also after treatment with an immune inducer such as DNFB (dinitrofluorobenzene). Thus, a 0.1% DNFB solution (hapten) was applied to both ears and left and right back skin 1 time per week for a total of 4 weeks.

Meanwhile, lactoferrin that alleviates inflammation of atopic dermatitis was administered to mice in a solution of 1. mu.g/ml in drinking water.

The experimental conditions of the mice were (1) hapten (-)/lactoferrin (-), (2) hapten (-)/lactoferrin (+), (3) hapten (+)/lactoferrin (-), (4) hapten (+)/lactoferrin (+), and 3 mice were used.

2. Extraction of protein from skin tissue of atopic dermatitis model mouse

Skin tissues of mice treated with hapten/lactoferrin for 4 weeks were cut, and the tissue pieces were cut with a knife and transferred to a centrifuge tube with a predetermined tare weight to measure the tissue weight. Tissue weight 0.85mg urea (urea), tissue weight 0.1. mu.l 1.5% SDS, tissue weight 0.1. mu.l 8.5% Triton X-100, tissue weight 0.05. mu.l 2ME buffer were added and homogenized with HG30 homogenizer (homogenerizer, HITACHI). After centrifugation at 15,000rpm (15,000Xg) at 10 ℃ for 30 minutes and recovery of the supernatant, the supernatant was centrifuged at 50,000rpm (100,000Xg) at 10 ℃ for 1 hour to obtain a sample. The amount of protein was quantified by dot blotting.

3. Extraction of proteins from skin tissue of atopic dermatitis patients

A cuticle tester [ asahi biomed co., Ltd (japanese original name "アサヒバイオメッド"), ]) was attached to a site where inflammation occurred (mainly the wrist) and a site near the site where inflammation did not occur in atopic dermatitis patients to obtain cuticle. As a control, samples were recovered from persons who had no atopic dermatitis. The corneometer, one site, 3 used sheets at the same site were recovered. Samples were scraped from the patches with a cell scraper in 50. mu.l of 1 Xsample buffer (samplebuffer) [83mM Tris-HCl (pH6.8), 2.7% SDS, 28% glycerol ]. After centrifugation at 15000rpm (15,000Xg) at 4 ℃ for 10 minutes, the supernatant was collected and the protein was quantified by the DC protein assay (BIO-RAD).

4. Two-dimensional gel electrophoresis (2-DE)

4-1 first dimension isoelectric point electrophoresis

60g of protein was mixed with a gel-swelling solution [5M urea (urea), 2M thiourea (thiourea), 0.5% ampholytes (pH3.5-10) (Amersham biosciences), 0.0025% orange G (orange G), 2.5mM TBP, and 1% Triton X-100 (Triton X-100) ] to make the total amount to 340. mu.l, and then swollen overnight at 20 ℃ with Immobiline Dry gel (Immobiline Dry-Strip gel) (18cm, pH3-10, NL) (Amersham biosciences). Using an apparatus of Anatech, isoelectric point electrophoresis was carried out in the first dimension by a procedure of 20 ℃ for 500V-2 hours, 700V-1 hours, 1000V-1 hours, 1500V-1 hours, 2000V-1 hours, 3000V-1 hours, 3500V-10 hours. After electrophoresis, the gel was immersed in SDS equilibration buffer [5.8M urea (urea), 0.06M thiourea (thiourea), 0.5% Dithiotheritol (DTT) (w/v), 25% glycerol (glycerol), 0.0025% BPB ] and equilibrated at room temperature for 1 hour.

4-2 second dimension SDS-PAGE

In the second dimension, SDS-PAGE was performed on an 18 cm. times.18 cm, 7.5% acrylamide gel using a buffer of Tris-Tris (hydroxymethyl) methylglycine (tricine) system [ cathode buffer: 0.05M Tris, 0.05M Tris (hydroxymethyl) methylglycine (tricine), 0.05% SDS, anode buffer: 1M Tris-HCl (pH8.8) ].

4-3 electrophoretic blotting

The gel after completion of SDS-PAGE was transferred with a constant current of 150mA on a PVDF membrane (ProBlottMembrans (Applied biosystems)) of 20 cm. times.20 cm using a semidry transfer apparatus (Nihon Eido Co., Ltd. (Japanese original name "Japan エイドー")) for 2 hours. As the transfer buffer, 0.3M Tris-HCl (pH10.4) and 20% methanol (methanol) were used as anolyte 1, 25mM Tris-HCl (pH10.4) and 20% methanol (methanol) as anolyte 2, 25mM Tris-HCl (pH10.4) and 20% methanol (methanol) as catholyte and 40mM 6-aminocaproic acid (6-amino hexanoic acid) were used as catholyte (Wako pure chemical industries). After the transfer, the sample was washed 3 times with TTBS buffer [20mM Tris-HCl (pH7.5) (BIO-RAD Co., Ltd.), 500mM NaCl, 0.3% Tween-20 (Tween-20) (BIO-RAD Co.) ] for 20 minutes, and then washed 3 times with MQW2 minutes. Then, the membrane is sealed and then is soaked in 50ml of Colloidal Gold solution [ Colloidal Gold Total Protein Stain (BIO-RAD corporation) ], and the Protein is dyed after 1-2 hours of oscillation. After the membrane dyeing, the colloidal gold solution was removed, washed with pure water for 1 minute and 5 times, and then dried.

5. Identification of proteins

5-1 reductive S-alkylation of proteins on PVDF membranes and protease cleavage

The protein spot transferred to the PVDF membrane was excised, placed in a test tube, and 100 to 300. mu.l of a buffer for reduction [8M guanidine hydrochloride (guanidine-HCl) (pH8.5) ], 0.5M Trisbase, 0.3% EDTA-2Na (w/v), 5% acetonitrile ] was added. To the reduction buffer, 1mg of DTT (dithioreitol) was added, the inside of the tube was purged with nitrogen, and the tube was allowed to stand at room temperature for 1 hour to reduce the protein. Before the reaction, iodoacetic Acid (Monoiodoacetic Acid) dissolved in 1M NaCl in an amount of 3mg was added, and the mixture was stirred for 15 to 20 minutes in the dark to perform S-carboxymethylation. After completion, the PVDF membrane was taken out, washed with pure water under stirring for 5 minutes, and then stirred in 2% acetonitrile in the same manner. The PVDF membrane was removed, transferred to a tube containing Lys-C enzyme digestion buffer [70% acetonitrile/20 mM Tris-HCl (pH9.0) ] and washed 2 to 3 times, and then completely immersed in the Lys-C enzyme digestion buffer for enzyme digestion for 1 hour.

5-2 Mass analysis and peptide Mass fingerprinting

The protease-cleaved solution was diluted 7-fold to make the acetonitrile concentration 10%. As a pretreatment, in order to activate the filler portion of the ZipTipc18 pipette tip (MILLIPORE corporation), 50% acetonitrile/0.1% TFA (trifluoroacetic acid: trifluo-acetic acid) was repeatedly aspirated and discharged in a repeated manner, and 2% acetonitrile/0.1% TFA (trifluoroacetic acid: trifluo-acetic acid) was repeatedly aspirated and discharged. Then, the protease cleavage solution was sucked and discharged several times by using an activated tip of a Zip tip 18 pipette, so that the fragmented peptides were adsorbed on the packed portion. Then, 2% acetonitrile/0.1% TFA (trifluoroacetic acid) was repeatedly aspirated and discharged to remove salts. 0.5 to 1. mu.l of a saturated base solution dissolved in 50% acetonitrile/0.1% TFA (trifluoroacetic acid) was aspirated, left for about 10 seconds, and dropped into a target probe attached to a mass spectrometer. The sample was allowed to dry and assayed by mass spectrometry (MALDI-TOF MS). Based on the obtained mass values, identification was performed with reference to proteins registered in a database (MS-Fit, Mascot Search).

Western blotting method

SDS-PAGE was performed by the Tris-Glycin system of Laemmli using the samples for Western blotting. After SDS-PAGE, the gel was run at 1cm intervals²A constant current of 0.8mA was transferred to PVDF membrane (MILIPORE Co.), and then blocked overnight at 4 ℃ using 5% skim milk/PBS (-). The PVDF membrane with 0.1% Tween20 (Tween 20)/PBS (-) washing 3 times, and 1 times of antibody at room temperature for 1 hours. Next, 2 times the antibody was diluted with biotinylated anti-goat IgG (Biotinylated anti-goat IgG) (Vector Laboratories Inc. [ 1: 1000 (dilution)]After shaking for 1 hour, avidin D conjugated to alkaline phosphatase (Vector Laboratories, Inc.) (1: 1000 dilution) was reacted for 1 hour with 5-bromo-4-chloro-3-indolyl phosphate (5-bromo-4-chloro-3-indolyl phosphate) at a concentration of 0.6mg/ml,1.2mg/ml nitrotetrazolium blue (nitrobluetetrazolium), 0.1M Tris-HCl (pH9.5), 5mM MgCl₂And (4) developing color. And 2 times the antibody was detected by fluorescent coloration using an Enhanced chemiluminescence (Enhanced chemilumine luminescence) (ECL) (Amersham biosciences) kit using horseradish peroxidase-labeled anti-mouse IgG (HRP labelled anti-mouse IgG) (Amersham biosciences) (1: 1000 dilution), horseradish peroxidase-labeled anti-rabbit IgG (HRP labelled anti-rabbit IgG) (Amersham biosciences) (1: 1000 dilution), horseradish peroxidase-labeled anti-rat IgG (HRP labelled-rat IgG) (Amersham biosciences) (1: 1000 dilution), and horseradish peroxidase-labeled anti-sheep IgG (HRP labelled anti-goat IgG) (Santa Cruz) (1: 1000 dilution). The kind and dilution of the 1-time antibody used were as shown in the following, mouse anti-HSP70monoclonal antibody (mouse anti-HSP70monoclonal antibody) (Santa Cruz Biotechnology Co.) (dilution 1: 1000), rabbit anti-annexin II polyclonal antibody (rabbit anti-annexin II polyclonal antibody) (Santa Cruz Biotechnology Co.) (dilution 1: 1000), mouse anti-HSP90monoclonal antibody (mouse anti-HSP90monoclonal antibody) (Santa Cruz Biotechnology Co.) (dilution 1: 1000), rat anti-GRP94 monoclonal antibody (rat anti-GRP94 monoclonal antibody) (Strophan sGEN.) (dilution 1: 1000), mouse anti-galectin-1monoclonal antibody (mouse anti-galectin-1monoclonal antibody) (Strophan Biotechnology R monoclonal antibody R) (Strophan sGEN Co.) (dilution 1: 1000)&D Systems Co.) (1: 1000 dilution), rat anti-galectin-3monoclonal antibody (rat anti-galectin-3monoclonal antibody) (R&D Systems Co.) (1: 1000 dilution), mouse anti-galectin-4monoclonal antibody (mouse anti-galectin-4monoclonal antibody) (R&D Systems Co.) (1: 1000 dilution), mouse anti-galectin-7monoclonal antibody (R)&DSystems Corp. (1: 1000 dilution), mouse anti-galectin-8monoclonal antibody (mouse anti-galectin-8monoclonal antibody) (R)&D Systems Co.) (1: 1000 dilution), goat anti-galectin-9polyclonal antibody (Santa Cruz Biotechnology Co.) (1: 1000 dilution), rabbitAnti-desmin polyclonal antibody (Santa Cruz Biotechnology Co.) (dilution 1: 1000), goat anti-vimentin polyclonal antibody (goat anti-vimentin polyclonal antibody) (Santa Cruz Biotechnology Co.) (dilution 1: 1000), rabbit anti-enolase-1polyclonal antibody (Rabbit anti-enolase-1polyclonal antibody) (Santa Cruz Biotechnology Co.) (dilution 1: 1000), rabbit anti-ematein/noggin/moesin polyclonal antibody (goat anti-ezrin/goat polyclonal antibody) (Chemicals Co.) (dilution 1: 1000), goat anti-FABP-4polyclonal antibody (goat anti-FABP-4polyclonal antibody) (goat anti-mouse monoclonal antibody A: 1000), goat anti-FABP-4polyclonal antibody (goat anti-mouse monoclonal antibody A: 1: 32) (dilution 1), Goat anti-apolipoprotein A1 (goat anti-apolipoprotein A1) (Abcom Co.) (1: 1000 dilution), goat anti-PARK7polyclonal antibody (goat anti-PARK7polyclonal antibody) (Abcom Co.) (1: 1000 dilution), mouse anti-Rho GDI monoclonal antibody (mouse anti-Rho GDI monoclonal antibody) (Santa Cruz Biotechnology Co.) (1: 1000 dilution), goat anti-FABP-5polyclonal antibody (goat anti-FABP-5polyclonal antibody) (R)&D Systems (1: 1000 dilution), mouse anti-SCCA2monoclonal antibody (mouse anti-SCCA2monoclonal antibody) (Santa Cruz Biotechnology) (1: 1000 dilution), rabbit anti-FABP-5polyclonal antibody (BioVendor) (1: 5000 dilution), rabbit anti-human albumin polyclonal antibody (Rabbit anti-human albumin polyclonal antibody) (Inter-Cell) (1: 1000 dilution).

7. Measurement of the degree of reduction in keratinocyte area

The cutin tester is pressed on the affected part to peel off the cuticle cells. The keratometer from which the stratum corneum cells were collected was stained with 1% brilliant green and 0.5% gentian violet aqueous solution, and observed using a digital microscope (VHX-100, KEYENCE Co, ltd., Japan) (modification Fuchi et al, JSCCJ, 23, 1, 1989. japanese prose: Fuchi ら modification を, JSCCJ, 23, 1, 1989.). The keratinocyte area of the resulting image was scored on a 5-point scale (1 point: small, 2 points: slightly small, 3 points: general, 4 points: slightly large, 5 points: large) by visual evaluation by a professional judge.

8. Determination of transdermal water loss

Measured using a commercially available skin moisture loss tester (Tewameter) (TEWAMETER TM210, manufactured by Courage + Khazaka electronic GmbH). The principle of measurement is to measure the vapor pressure at 2 points of several mm on the skin and calculate the amount of water evaporated from the epidermis, assuming that the water lost from the skin surface into the air is diffused according to Fick's rule.

9. Determination of marker proteins by ELISA (enzyme-Linked immunosorbent assay)

In a 96-well ELISA plate, a stratum corneum protein solution diluted to 0.2. mu.l/. mu.l with Phosphate Buffered Saline (PBS) was added at 50. mu.l/well and adsorbed at 4 ℃ for 18 hours. After removing the stratum corneum protein solution, the membrane was immersed in a blocking solution { PBS containing 1% Bovine Serum Albumin (BSA) }, and blocked at 37 ℃ for 1 hour. After washing with a washing solution { PBS containing 0.05% polyoxyethylene (20) sorbitan monolaurate (Wako pure chemical industries, Ltd.) }, 1 time of an antibody solution { each antibody prepared at 5mg/ml with a washing solution } was added to each of the wells at 50. mu.l/well, and the reaction was carried out at 37 ℃ for 2 hours. After washing, 2 times of the antibody solution { HRP (horseradish peroxidase: Horseradi peroxidase) -converted anti-mouse immunoglobulin G (VECTOR LABORATORIES) prepared at 1mg/ml with a washing solution or HRP (horseradish peroxidase: Horseradi peroxidase) -converted anti-rabbit immunoglobulin G (VECTOR LABORATORIES) prepared at 1mg/ml with a washing solution was added at 50. mu.l/well, respectively, and reacted at 37 ℃ for 1 hour. After washing, chemiluminescence reaction was performed using an Enhanced Chemiluminescence (ECL) kit, and detection was performed by a chemiluminescence detector (spectra max Lmax ii 384, Molecular devices) to quantify the amount of the product.

Results of the experiment

1. Hapten treatment of atopic dermatitis model mice

The atopic dermatitis model mice used in this study (NC/Nga mice) were haptens under conventional circumstances

When the animals were raised after the treatment, the onset of dermatitis that closely resembles atopic dermatitis was visually confirmed 4 weeks after the hapten treatment. In this study, lactoferrin (a protein having a molecular weight of 8 ten thousand and binding to iron ions contained in saliva or blood) having an effect on atopic dermatitis was mixed into drinking water and drunk. As a result of raising the mice after the hapten and lactoferrin treatments, inflammation similar to human atopic dermatitis was observed on the backs of the mice treated with the hapten coatings, and the mice further drunk water mixed with lactoferrin after the hapten coatings had suppressed inflammation compared with the mice treated with the hapten coatings alone (fig. 1). When a protein whose expression is changed by hapten treatment accompanying development of atopic dermatitis is treated with lactoferrin, the symptoms of atopic dermatitis are alleviated and the level of atopic dermatitis is restored to a level equivalent to that in the absence of treatment, and the protein is considered to be a promising marker.

2. Two-dimensional electrophoretic analysis Using skin tissue of atopic dermatitis model mouse

To identify proteins associated with atopic dermatitis, the skin of mice, whether hapten-coated or untreated, lactoferrin-treated or untreated, was analyzed for altered proteins using two-dimensional electrophoresis.

The skin (site of inflammation at the time of hapten application) of a mouse not treated (control), a mouse coated with hapten, a mouse drinking lactoferrin as a control, and a mouse further drinking lactoferrin after hapten application were taken out, a sample buffer was added by weight, and after the sample buffer was crushed with a Polytron type homogenizer, the crushed sample was centrifuged at 15000rpm (15,000Xg) for 30 minutes, and then the supernatant was collected. The supernatant was ultracentrifuged at 100,000Xg for 1 hour, and the supernatant was used as a sample. Two-dimensional electrophoresis was performed on a gel strip (strip gel) with pH3-10 in the first dimension and on a 7.5% acrylamide gel in the second dimension. The altered protein was detected by staining with a colloidal gold solution, and the protein in which the alteration was observed was subjected to molecular characterization using a mass spectrometer (MALDI-TOF MS) (FIG. 2(a) (b)). As a result, 43 proteins were identified among the 49 proteins analyzed. Among the identified proteins, proteins whose expression was increased in hapten-treated mice (mice producing atopic dermatitis inflammation) were identified as FABP-5, Apolipoprotein A1(Apolipoprotein A1), Vimentin (Vimentin), and the like, and 4 unknown proteins were identified. On the other hand, in the hapten-treated mice, proteins whose expression was decreased were identified as Galectin-3 (Galectin-3), Desmin (Desmin), PARK7 and the like. And the majority of the identified proteins are the framework proteins of keratin 5 (kerat in 5), keratin16 (kerat 16), Desmin (Desmin), Vimentin (Vimentin), Moesin (Moesin), etc.

3. Confirmation Using antibodies to proteins altered in atopic dermatitis model mice

In order to confirm the expression of the proteins shown above which are altered by the onset of atopic dermatitis, analysis was performed by Western blotting using various antibodies. First, since proteins identified from the results of two-dimensional electrophoresis contain Galectin-3 (Galectin-3), they were confirmed focusing on the Galectin family. Galectins are a kind of glycoprotein and do not have a normal signal sequence, but are released extracellularly with or without stimulation such as an immune response. It has been reported that each of the galectin molecules shows different tissue distribution and is involved in various physiological phenomena such as immunomodulation, cell-matrix adhesion, intercellular adhesion, wound healing and the like (refer to J.biol.chem.264:1310-1316(1989), J.biochem.104:1-4(1988), Proc.Natl Acad.Sci.USA.87:7324-7328(1990), Eur.J.biochem.248:225-230(1997), Dev.biol.168:259-271(1995), J.biol.chem.270:5823-5829(1995), and Proc.Natl.Acad.Sci.U.S.A.93:7252-7257 (1996)).

Therefore, changes in expression levels in the skin of mice of the atopic dermatitis model were analyzed using antibodies to Galectin-1, -3, -4, -7, -8, -9(Galectin-1, -3, -4, -7, -8, -9) contained in the Galectin (Galectin) family, in addition to Galectin-3 (Galectin-3) (FIG. 3). As a result, Galectin-1, -3, -4, -7, -8(Galectin-1, -3, -4, -7, -8) were expressed in a reduced amount in the hapten-treated mice as compared with the untreated mice (control mice). On the other hand, it was also found that, in the Galectin-1, -4, -7, -8(Galectin-1, -4, -7, -8), even in the hapten-treated mice, the expression level of the mice was restored to a level close to that of the hapten-untreated mice (control mice) after drinking lactoferrin. Galectin-9 (Galectin-9) is not changed by hapten treatment, and thus it is considered to have a low correlation with atopic dermatitis.

The same analysis was performed for cytoskeletal protein Desmin (Desmin), Moesin/Ezrin (Ezrin)/radxin (radiaxin), and Vimentin (Vimentin) (fig. 4). The hapten-treated mice showed a decrease in Desmin (Desmin) expression and an increase in Moesin (Moesin)/Ezrin (Ezrin)/radxin (radiaxin) (50KDa cleaved form) and Vimentin (Vimentin) expression compared to untreated control mice. In particular, the low molecular weight cleavage pattern of Moesin/Ezrin/Radixin was significantly increased by hapten treatment and was not decreased by lactoferrin administration. And the Moesin/Ezrin/Radixin protein belonging to the same family was identified as Moesin by two-dimensional electrophoretic analysis. Therefore, the antibody used twice was an antibody recognizing these 3 kinds, and it is considered that due to inflammation, Moesin (Moesin) or other proteins are highly expressed alone or in many cases.

With respect to heat shock proteins HSP70, HSP90, GRP94, the hapten-treated mice had reduced expression levels of HSP70 and HSP90 compared to untreated mice (control mice), but expression of GRP94 was unchanged regardless of hapten treatment and no treatment (fig. 4).

Also, FABP-4 (fatty acid binding protein-4: fat acid binding protein-4), FABP-5 (fatty acid binding protein-5: fat acid binding protein-5), Enolase 1(Enolase1), PARK7(DJ-1), annexin II (annexin II), Apolipoprotein A1(Apolipoprotein A1), and Western GDI were analyzed by the blotting method (FIG. 5). As a result, the expression level of Annexin II (Annexin II), Enolase 1(Enolase1), FABP-4, and PARK7 was reduced in the hapten-treated mice as compared with the untreated mice (control mice). In addition, even in the hapten-treated mice except FABP-4, the expression level of the mice was restored to a level close to that of the hapten-untreated mice (control mice) after drinking lactoferrin. On the other hand, for Rho GDI, FABP-5, Apolipoprotein A1(Apolipoprotein A1), the expression was increased in hapten-treated mice compared to untreated mice (control mice) (FIG. 5). Rho GDI is known to reduce intercellular adhesion, so that increased expression of this protein may be associated with its involvement in skin detachment in atopic dermatitis due to atopic dermatitis.

4. SDS-PAGE analysis using skin tissue from atopic dermatitis patients

From the above results, candidate atopic dermatitis markers using an atopic dermatitis model mouse were screened, but whether these proteins could be markers of human atopic dermatitis was investigated. Therefore, samples were actually taken from the inflammatory site (fig. 6, a.p.) of the atopic dermatitis-affected person and the non-inflammatory site (Control), and the same site (Control) of the volunteer who did not develop atopic dermatitis using a sheet-like article called a cuticle tester. Samples of 3 patches were recovered from the same site of human skin, and then skin tissue was lysed using 1 x SDS sample buffer. Thereafter, SDS-PAGE was performed using samples of atopic dermatitis-affected persons (2 persons) and non-affected persons (3 persons), and proteins whose expression was increased in the atopic dermatitis-affected samples were identified using a mass spectrometer (MALDITOF-MS). As a result, it was revealed that the expression of Annexin II (Annexin II), squamous cell carcinoma antigen 1 (squamous cell carcinomaantigen-1) (SCCA1), squamous cell carcinoma antigen2 (squamous cell carcinomaantigen-2) (SCCA2), fatty acid binding protein-5(FABP-5), Serum albumin (Serum albumin) and immunoglobulin G (immunoglobulin G) increased with the onset of atopic dermatitis (FIG. 6). On the other hand, it was also shown that the expression of arginase I (ArginaseI) and Uracil DNA glycosylase (Uracil-DNAycosylase) was reduced in atopic dermatitis patients (FIG. 6).

5. Analysis by Using various antibodies against skin tissue of atopic dermatitis patients

When analyzed by Western blotting using atopic dermatitis model mice, 17 proteins were found to be changed. These proteins were tested for the same changes in the onset of inflammation in atopic dermatitis in humans (FIG. 7). Meanwhile, proteins that change were examined by SDS-PAGE analysis using skin tissues of atopic dermatitis patients.

For Annexin II (Annexin II), the sites of onset (fig. 7, a.p.) were increased in expression compared to the non-onset sites (controls) in atopic dermatitis patients, and the molecular weight was slightly lower than that of the bands in atopic dermatitis non-onset patients. The reason for this is not clear, but it is considered that this difference in molecular level may be related to the difference in constitution between atopic dermatitis-affected and non-affected persons.

For Enolase 1(Enolase1), squamous cell carcinoma antigen2 (squamous cellcercinoma antigen 2) (SCCA2), increased at the sites of onset in1 atopic dermatitis patient.

Although PARK7 showed no change in expression depending on the patients with atopic dermatitis, all of the patients with atopic dermatitis showed a decrease in expression compared to the patients with non-atopic dermatitis.

In addition, for Apolipoprotein a1(Apolipoprotein a1), the non-diseased site of atopic dermatitis and the non-diseased site of atopic dermatitis patients were not expressed at all, and the expression was increased only at the diseased site of atopic dermatitis.

The above results are summarized in Table 1.

TABLE 1

6. Analysis by Using various antibodies to skin tissue of a patient

In order to confirm the utility of atopic dermatitis marker candidates detected from skin tissues of atopic dermatitis model mice and a few atopic dermatitis patients, the correlation between the severity of atopic dermatitis and the severity of atopic dermatitis was measured using skin samples of 17 (7 males, 10 females) and 15 (10 males, 5 females) non-diseased volunteers of atopic dermatitis patients who were diagnosed by a dermatologist. The patients with atopic dermatitis were classified according to the severity (revised edition of japanese society for skin science, atopic dermatitis treatment guideline 2004, [ japanese original name: "japanese society for skin , アトピー for skin science, ガイドライン 2004, modified version ]), with 1 having a severity of 1, 7 having a severity of 2,6 having a severity of 3, and 3 having a severity of 4. From 17 atopic dermatitis volunteers, data on eosinophils, IgE, LDH, etc. in blood, which are known to be associated with the severity of atopic dermatitis, were collected. Skin samples were collected with a cuticle tester and analyzed for expression of 6 proteins by Western blotting (fig. 8(a) (b)).

Enolase 1(Enolase1) could not be detected from the skin of non-atopic dermatitis patients, but was found to be expressed in large amounts in the skin of atopic dermatitis patients. Among patients, there are 12 out of 17 patients expressing more inflammatory sites than non-inflammatory sites. In contrast, there were 2 patients who were reduced.

Fatty acid binding protein-5(FABP-5) was not detected in the skin of non-affected persons, but was strongly expressed at the inflammatory sites of atopic dermatitis patients, although it was not detected in1 person. There were 13 of 17 patients expressing more at the site of inflammation than at the site of non-inflammation.

For Squamous cell carcinoma antigen2 (Squamous cell carcinoma antigen 2) (SCCA2), 1 was weakly expressed in non-sick but the expression was very significantly increased in the skin of atopic dermatitis patients. Among 17 patients expressing much at the site of inflammation, 9 patients were present, whereas 1 patient was present in the decreased number. A deviation in expression was observed compared with FABP-5.

For apolipoprotein a1(apolipoprotein a1), 1 person was weakly expressed in the skin of non-diseased persons, and a tendency of strong expression was detected in specific patients among atopic dermatitis patients. Among 17 patients with high expression in inflammatory parts, 9 patients were present, and 1 patient was present in a reduced number.

Serum albumin (Serum albumin) was strongly expressed in all atopic dermatitis patients, and the difference between all inflammatory and non-inflammatory parts was small. Among non-affected patients, the expression was weak except for 1.

For Annexin II (Annexin II), although expression was weak, a large amount of expression was detected in the skin of atopic dermatitis patients, and almost no expression was detected in non-diseased patients. There were 6 of 17 patients with high expression in the inflammatory part compared to the non-inflammatory part, and there were 6 patients with decreased expression.

In addition to Annexin II (Annexin II) having a low expression intensity, the expression intensity of 5 samples of each marker protein, Enolase 1(Enolase1), FABP-5, SCCA2, Apolipoprotein A1(Apolipoprotein A1), and serum albumin (Serumalbumin) was quantified. The results were summarized according to the severity of the patients, as shown in FIGS. 9(a) and (b).

FABP-5, Serum albumin (Serum albumin), and Enolase 1(Enolase1) show a high correlation with the severity of atopic dermatitis of the skin of a patient, and therefore can be used to determine the severity of atopic dermatitis of a patient. Serum albumin (serum albumin) can be detected even from non-inflammatory sites, and therefore it reflects the constitution of patients associated with the onset of atopic dermatitis. Apolipoprotein A1(Apolipoprotein A1) and SCCA2 are biased in expression, and Annexin II (Annexin II) is characterized by high expression at non-inflammatory sites. The expression of these markers may also reflect the patient's characteristics.

The results are summarized in Table 2.

TABLE 2

7. Validation of marker effectiveness in persons with a history of atopic dermatitis

The number of people who experience hospital frequently due to atopic dermatitis is about 634 ten thousand, and the number of people who present allergic skin with atopic dermatitis symptoms is presumed to be about 1200 thousand. Atopic dermatitis is also very effective not only in the treatment by a medicament but also in the skin care by a moisturizing agent (fieldish, Fragrance Journal, p13-19, month 6 2003), and people who care skin with medical and health products and cosmetics are also very popular. From the above, it is considered that the use of the marker found in the present study is useful not only for the diagnosis of atopic dermatitis by a dermatologist but also for the skin diagnosis in a counseling store and the skin diagnosis at home. Therefore, the utility of the atopic dermatitis marker candidate was confirmed for a human having a history of atopic dermatitis that had been once developed and experienced by a dermatologist.

As characteristic changes of atopic dermatitis, there are clearly an increase in percutaneous water loss (TEWL) due to a decrease in the barrier function of the stratum corneum, a decrease in the area of keratinocytes due to insufficiency of keratinization, and the presence of nucleated cells in the cutin (Tagami h.et al, j.invest.dermaltol.symp.proc., 6, 1, 87 to 94, 2001). Therefore, the effectiveness of the candidate marker was examined by using the degree of increase in TEWL and the degree of reduction in keratinocyte area as indicators of the degree of atopic dermatitis inflammation.

Evaluation was performed on 11 healthy subjects and 10 volunteers who had a history of atopic dermatitis. The correlation between the degree of development of TEWL and the reduction in keratinocyte area and the expression levels of 6 candidate markers [ FABP-5, Galectin-7 (Galectin-7), Enolase 1(Enolase1), SCCA2, Apolipoprotein a1(Apolipoprotein a1), and Annexin II (Annexin II) ] was determined using the inner upper arm part of the normal part of healthy persons and persons having a history of atopic dermatitis, and using the inflammation parts (7 inner elbow parts, 1 neck part, 1 finger suture part, and 1 knee inner part) of persons having a history of atopic dermatitis. The degree of reduction in the keratinocyte area was measured by a peel-off cuticle staining method, and the degree of increase in TEWL was measured by a skin moisture loss meter (Tewameter). The expression levels of 6 candidate markers were measured by ELISA method using the stratum corneum of healthy subjects and persons with a history of atopic dermatitis, which were collected by a keratometer, and proteins extracted therefrom.

The results of determining the correlation between the degree of hyperactivity of TEWL and the expression degrees of 6 candidate markers [ FABP-5, Galectin-7 (Galectin-7), Enolase1, SCCA2, Apolipoprotein A1(Apolipoprotein A1), Annexin II (Annexin II) ] are shown in FIG. 10. TEWL shown on the X-axis of fig. 10 was almost equal in the normal sites of healthy persons and persons with a history of atopic dermatitis, but showed an increase in the inflammatory sites of persons with a history of atopic dermatitis. The expression of 3 markers FABP-5, Galectin-7 (Galectin-7) and Enolase 1(Enolase1) on the Y axes in FIGS. 10(a) to (c) was associated with increased TEWL in inflammatory sites of people with a history of atopic dermatitis compared with normal sites of healthy people and people with a history of atopic dermatitis. On the other hand, the expression levels of 3 markers SCCA2, apolipoprotein a1(ApolipoproteinA1), and Annexin II (Annexin II) shown in the Y axes of fig. 10(d) to (f) are effective for diagnosing the risk of developing atopic dermatitis, in healthy subjects < normal sites in people with a history of atopic dermatitis < inflammatory sites in people with a history of atopic dermatitis.

The results of measuring the correlation between the degree of reduction in keratinocyte area and the expression degrees of 6 candidate markers [ FABP-5, Galectin-7, Enolase 1(Enolase1), SCCA2, Apolipoprotein A1(Apolipoprotein A1), Annexin II (Annexin II) ] are shown in FIG. 11. The keratinocyte area shown by the X-axis in fig. 11 is substantially equal in the normal parts of healthy persons and persons having a history of atopic dermatitis, but is decreased in the inflammatory parts of persons having a history of atopic dermatitis. The expression levels of 3 markers, FABP-5, Galectin-7 (Galectin-7) and Enolase 1(Enolase1), indicated by the Y axes in FIGS. 11(a) to (c), were increased in inflammatory sites of healthy subjects and subjects with a history of atopic dermatitis compared to normal sites of healthy subjects and subjects with a history of atopic dermatitis, with respect to the reduction in keratinocyte area. On the other hand, the expression levels of 3 markers SCCA2, Apolipoprotein a1(Apolipoprotein a1), and Annexin II (Annexin II) shown in the Y axes of fig. 11(d) to (f) are effective for diagnosing the risk of developing atopic dermatitis in healthy subjects < normal sites in persons with a history of atopic dermatitis < inflammatory sites in persons with a history of atopic dermatitis.

The present invention provides a protein whose expression is increased and a protein whose expression is decreased depending on the degree of inflammation of atopic dermatitis and the risk of onset of atopic dermatitis. By detecting the expression changes of these proteins, more accurate diagnosis and determination of the risk of developing atopic dermatitis can be performed based on the relationship with the cause and symptoms of atopic dermatitis. And these markers can be used for the development of therapeutic drugs for atopic dermatitis, cosmetics for sensitive skin, health foods, and the like.

Claims

1. Use of an antibody specifically recognizing a specific protein whose expression is changed accompanying inflammation of atopic dermatitis, said specific protein comprising galectin-7, for the preparation of a preparation for diagnosing or detecting atopic dermatitis.