JP2023096623A - Method for evaluating or selecting agent for preventing or improving vitiligo - Google Patents

Abstract

To provide a method for evaluating or selecting an agent for preventing or improving vitiligo.SOLUTION: Provided is a method for evaluating or selecting an agent for preventing or improving vitiligo, comprising the following steps (1) to (3): (1) bringing a test substance into contact with a tissue or cell capable of expressing MMP-2; (2) measuring expression or activity of the MMP-2 in the tissue or cell; (3) based on a result of measurement in (2), evaluating or selecting the test substance that decreases the expression or activity of the MMP-2, as the agent for preventing or improving vitiligo.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a method for evaluating or selecting an agent for preventing or improving vitiligo.

Vitiligo is a condition in which the skin becomes depigmented due to a lack of skin melanocytes. The etiology of vitiligo is known to include genetic factors, autoimmune factors, induction by chemical substances such as phenol derivatives and catechol derivatives, but the onset and pathogenesis mechanisms of vitiligo are still unknown.
Common treatments for vitiligo include topical corticosteroids, topical calcineurin inhibitors (tacrolimus and pimecrolimus), and ultraviolet (UVB) therapy. However, none of them are reliable treatments, and vitiligo is regarded as one of the intractable diseases for which there is no definitive treatment.

Matrix metalloproteinase (MMP) is a degrading enzyme that degrades proteins such as collagen, which is an extracellular matrix component. While MMPs are required for the physiological maintenance and remodeling of the extracellular matrix, they are essential enzymes for a variety of normal physiological processes such as embryogenesis, morphogenesis, reproduction, tissue resorption and tissue remodeling. , inflammation, arthritis, cardiovascular disease, fibrosis and cancer. At present, more than 20 kinds of human MMPs are known, and they are classified into collagenase, gelatinase, stromelysin, membrane-type MMP, matrilysin, etc. according to the domain structure.

Recently, it has been reported that MMP-9 in epidermal cells is involved in the adhesion of epidermal cells and melanocytes, and that MMP-9 inhibition may suppress the detachment of melanocytes from the epidermal basement membrane and suppress the loss of melanocytes in vitiligo. (Non-Patent Document 1 and Patent Document 1). On the other hand, it has been reported that no change was observed in the expression of MMP-2 in serum samples (Non-Patent Document 1).

Japanese Patent Publication No. 2020-504719

2020 June 4; 5(11): e133772

The present invention relates to providing a method for evaluating or selecting a preventive or ameliorating agent for vitiligo.

The inventors of the present invention focused on the skin basement membrane (BM) existing between the epidermis and dermis, and found that the structure of the skin basement membrane is abnormal in vitiligo lesions. The cause was found to be overexpression of MMP-2 in dermal fibroblasts in vitiligo lesions. They also found that by using the expression of MMP-2, whose production is enhanced in vitiligo lesions, and TIMP-2, which is an inhibitor thereof, as indicators, it is possible to search for or evaluate agents for the prevention or improvement of vitiligo. rice field.

That is, the present invention relates to the following 1) and 2).
1) A method for evaluating or selecting an agent for preventing or improving vitiligo, comprising the following steps (1) to (3).
(1) Step of contacting a test substance with a tissue or cell capable of expressing MMP-2 (2) Step of measuring the expression or activity of MMP-2 in the tissue or cell (3) Measured in (2) Step 2) evaluating or selecting a test substance that reduces MMP-2 expression or activity as a vitiligo preventive or ameliorating agent based on the results, including steps (4) to (6) below, vitiligo prevention or Method for evaluating or selecting improvers.
(4) Step of contacting a test substance with a tissue or cell capable of expressing TIMP-2 (5) Step of measuring the expression of TIMP-2 in the tissue or cell A step of evaluating or selecting a test substance that increases the expression of TIMP-2 as a vitiligo preventive or ameliorating agent based on

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to search for or evaluate a vitiligo preventive or ameliorating agent with a new mechanism for preventing or ameliorating vitiligo.

FIG. 3 is a diagram showing the results of localized observation of skin basement membrane constituent molecules (type IV collagen) by immunoelectron microscopy. FIG. 4 shows MMP-2 expression in dermal fibroblasts of lesions of healthy subjects and vitiligo patients. FIG. 2 shows abnormalities in the skin basement membrane and disappearance of melanocytes in a three-dimensional skin model introduced with MMP-2 overexpressing human fibroblasts. (A) Model construction diagram, (B) Observation diagram of depigmentation-like phenomenon, (C) Immunostaining microscope image. FIG. 2 shows abnormalities in the skin basement membrane and disappearance of melanocytes in a human skin tissue culture system introduced with MMP-2 overexpressing human fibroblasts. FIG. 2 shows depigmentation in mouse skin dermis introduced with MMP-2 overexpressing mouse fibroblasts.

As used herein, MMP-2 (matrix metalloproteinase-2) is an MMP classified as a gelatinase and is also called gelatinase A. MMP-2 is an enzyme that degrades extracellular matrices such as gelatin, type IV, V, VII, X, and XI collagen, fibronectin, elastin, and proteoglycan, and mainly degrades basement membrane components. Overexpression and activation of MMP-2 are associated with basement membrane disruption and cancer invasion (Quintero-Fabian, S. et al. Role of Matrix Metalloproteinases in Angiogenesis and Cancer. Frontiers in Oncology 9, doi:10.3389/fonc.2019.01370 (2019)).

TIMPs (tissue inhibitors of metalloproteinases) are endogenous inhibitors of MMPs and regulate the balance between extracellular matrix degradation and synthesis by MMPs. TIMP-2 forms a complex with active MMP-2 and suppresses the activity of MMP-2 (Arpino, V., Brock, M. & Gill, S. E. The role of TIMPs in regulation of extracellular matrix proteolysis. Matrix Biology 44-46, 247-254, doi: https://doi.org/10.1016/j.matbio.2015.03.005 (2015)).

The skin basement membrane is a thin membrane-like extracellular matrix that exists between the epidermis and the dermis. The skin basement membrane is mainly composed of type IV collagen and laminin. The function of the skin basement membrane is to maintain the functions of cells and the structure of the dermis, act as a scaffold for epidermal keratinocytes (keratinocytes) and pigment cells (melanocytes), control cell metabolism, and control cell polarity. There is maintenance, etc., and it plays an important role in supporting the skin.

Vitiligo is a condition in which the skin becomes depigmented due to a lack of skin melanocytes. Vitiligo vulgaris, in which depigmentation is caused by acquired melanocytes decreasing or disappearing, and chemical substance-induced depigmentation caused by chemical substances. Such chemical substances include raspberry ketone, rhododenol (4-(4-hydroxyphenyl)-2-butanol) which is a reduced form of raspberry ketone, phenol derivatives such as hydroquinone monobenzyl ether, catechol derivatives such as 4-tert-butylcatechol, and the like. is mentioned.

As shown in Examples below, as a result of localized observation of skin basement membrane constituent molecules (type IV collagen) by immunoelectron microscopy, the distribution of skin basement membrane constituent molecules was uneven in vitiligo lesions (Fig. 1). In addition, MMP-2 was overexpressed in the dermis of vitiligo lesions (Fig. 2). In addition, the expression of MMP-9 was lower than that of MMP-2. Furthermore, in the three-dimensional skin model and human skin tissue culture system introduced with MMP-2 overexpressing human fibroblasts, loss of melanocytes was observed (FIGS. 3 and 4), and MMP-2 overexpressing mouse fibroblasts were introduced. Formation of depigmentation was observed in the dermis of the mouse skin treated with this method (Fig. 5).
These results indicate that the structure of the skin basement membrane is abnormal in vitiligo lesions, and that the cause is overexpression of MMP-2 in dermal fibroblasts in vitiligo lesions. . Therefore, the regulation of MMP-2, whose expression is enhanced in vitiligo lesions, can be a target for the prevention or treatment of vitiligo. can be evaluated or selected for prevention or amelioration of

The method for evaluating or selecting an agent for preventing or improving vitiligo of the present invention includes the following steps (1) to (3).
(1) Step of contacting a test substance with a tissue or cell capable of expressing MMP-2 (2) Step of measuring the expression or activity of MMP-2 in the tissue or cell (3) Measured in (2) Based on the results, the step of evaluating or selecting a test substance that reduces the expression or activity of MMP-2 as an agent for preventing or improving vitiligo

In addition, the method for evaluating or selecting an agent for preventing or improving vitiligo of the present invention includes the following steps (4) to (6).
(4) Step of contacting a test substance with a tissue or cell capable of expressing TIMP-2 (5) Step of measuring the expression of TIMP-2 in the tissue or cell A step of evaluating or selecting a test substance that increases the expression of TIMP-2 as a vitiligo preventive or ameliorating agent based on

Tissues or cells capable of expressing MMP-2 and tissues or cells capable of expressing TIMP-2 include tissues that naturally have the MMP-2 gene or TIMP-2 gene and have the ability to express it. or cells, and tissues or cells into which the MMP-2 gene or TIMP-2 gene has been exogenously introduced so that it can be expressed. The tissue or cells may be tissues or cells collected from a living body, or cultured cells.
Tissues or cells that naturally have the MMP-2 gene or TIMP-2 gene and have the ability to express it include all tissues or cells of a living body, preferably skin taken from a mammal. Tissues or cells, for example, epidermal tissue, epidermal tissue-derived cells (epidermal keratinocytes, etc.), dermal tissue, dermal tissue-derived cells (fibroblasts, etc.), cultured cells derived therefrom, and the like. Preferred are dermal tissue and dermal tissue-derived cells (such as fibroblasts), and more preferred are dermal tissue-derived cells (such as fibroblasts).
Examples of mammals include humans, mice, rats, hamsters, rabbits, pigs, monkeys and the like. Humans are preferred.

Tissues or cells exogenously introduced to express the MMP-2 gene or TIMP-2 gene are, for example, by introducing a gene encoding MMP-2 or TIMP-2 into any mammalian tissue or cell. , by transforming the tissue or cells to express MMP-2 or TIMP-2, or to enhance expression of MMP-2 or TIMP-2.
Also, cells introduced with a construct in which a reporter gene such as luciferase is fused downstream of the promoter region of the MMP-2 gene or TIMP-2 gene can be used. Methods for introducing genes and constructs into cells include, but are not limited to, vector introduction by electroporation, lipofection, and the like.

As the cultured cells, three-dimensionally cultured skin cells are also preferably used. Specifically, EpiDerm TM (manufactured by MatTek Corporation), EpiSkin (manufactured by SkinEthic), RHE (manufactured by SkinEthic), Labcyte Epimodel (J-TEC company) and the like.

The test substance is not particularly limited as long as it can be used as a preventive or ameliorating agent for vitiligo, for example, animals, plants, marine organisms, microorganisms, etc. and extracts thereof; synthetic compounds; and mixtures and compositions thereof.

The means for contacting the test substance with the tissue or cells may be any means known in the art, for example, addition of the test substance to the tissue or cell culture medium, direct addition to the tissue or cells ( For example, dripping, coating, spraying, spraying, patching, etc.) can be mentioned.
The concentration and contact amount of the test substance may be appropriately determined based on the form, chemical properties, cytotoxicity, etc. of the test substance. For example, a predetermined amount of a test substance diluted to an appropriate concentration is exposed to dermal fibroblasts or the like under conditions of 25-37° C. and 5% CO ₂ for 24-48 hours.

The expression or activity of MMP-2 and the expression of TIMP-2 in said tissue or cell can be measured according to methods known in the art. The expression of MMP-2 and TIMP-2 can be measured using protein expression, mRNA expression, promoter activation, or the like as an index.

Examples of methods for measuring protein expression include immunohistochemistry, ELISA, Western blot, immunoprecipitation, mass spectrometry, etc., and combinations thereof.
Methods for measuring mRNA expression include dot blotting, Northern blotting, RT-PCR, real-time RT-PCR, microarrays, RNA sequencing, etc., and combinations thereof.
Methods for measuring promoter activity include fluorescent/optical measurement of promoter activity and transcription activity using a reporter gene (reporter assay).

MMP-2 activity can be measured, for example, by Western blotting, zymography, or detection of activated protein using a commercially available ELISA kit.

Then, based on the results measured as described above, a test substance that reduces the expression or activity of MMP-2 is evaluated or selected as an agent for preventing or improving vitiligo. Alternatively, a test substance that increases the expression of TIMP-2 is evaluated or selected as an agent for preventing or improving vitiligo.
Such evaluation or selection is performed, for example, before and after addition of the test substance, or by comparing a test substance-added group with a test substance-free group or a control substance-added group. Alternatively, evaluation is performed by comparing measurements between different concentrations of the test substance.

For example, if the expression or activity of MMP-2 in the test substance addition group is lower than that before the test substance addition, the test substance non-addition group, or the control substance addition group, the test substance is evaluated as an agent for preventing or improving vitiligo. or select. In this case, the determination can be made by determining whether the expression or activity of MMP-2 in the test substance added group is statistically significantly lower than the test substance added group, the test substance non-added group, or the control substance added group. can. Alternatively, when the expression or activity of MMP-2 in the test substance non-addition group or the control substance addition group is set to 100% before the test substance addition, the MMP-2 expression or activity in the test substance addition group is below a certain level, for example , 90% or less, preferably 70% or less, more preferably 50% or less.

In addition, if the expression of TIMP-2 is higher in the test substance addition group than before the test substance addition, in the test substance non-addition group, or in the control substance addition group, the test substance is evaluated or selected as an agent for preventing or improving vitiligo. do. In this case, it can be determined by whether or not the expression of TIMP-2 in the test substance addition group is statistically significantly higher than the test substance addition group, the test substance non-addition group, or the control substance addition group. Alternatively, when the expression of TIMP-2 in the test substance non-addition group or the control substance addition group before addition of the test substance is taken as 100%, the expression of TIMP-2 in the test substance addition group is above a certain level, for example, 110% or more. , preferably 130% or more, more preferably 150%.

The vitiligo preventive or ameliorating agent thus obtained can be used for preventive treatment of vitiligo by blending it in cosmetics, quasi-drugs, pharmaceuticals and the like.

The present invention further discloses the following aspects regarding the above-described embodiments.

<1> A method for evaluating or selecting an agent for preventing or improving vitiligo, comprising the steps of (1) to (3) below.
(1) Step of contacting a test substance with a tissue or cell capable of expressing MMP-2 (2) Step of measuring the expression or activity of MMP-2 in the tissue or cell (3) Measured in (2) Based on the results, the step of evaluating or selecting a test substance that reduces the expression or activity of MMP-2 as an agent for preventing or improving vitiligo

<2> A method for evaluating or selecting an agent for preventing or improving vitiligo, comprising the steps of (4) to (6) below.
(4) Step of contacting a test substance with a tissue or cell capable of expressing TIMP-2 (5) Step of measuring the expression of TIMP-2 in the tissue or cell A step of evaluating or selecting a test substance that increases the expression of TIMP-2 as a vitiligo preventive or ameliorating agent based on

<3> The method according to <1> or <2>, wherein vitiligo is preferably vitiligo vulgaris or chemical substance-induced depigmentation.
<4> The chemical-induced depigmentation is preferably chemical-induced depigmentation induced by a phenol derivative or a catechol derivative, more preferably chemical-induced depigmentation induced by rhododenol. <3> The method described.
<5> Tissues or cells capable of expressing MMP-2 or tissues or cells capable of expressing TIMP-2 are preferably mammalian epidermal tissue, epidermal tissue-derived cells, dermal tissue, dermal tissue-derived cells, or cultured cells derived therefrom, more preferably dermal tissue or dermal tissue-derived cells, more preferably dermal tissue-derived cells, according to any one of <1> to <4>.
<6> The method according to <5>, wherein the mammal is preferably a human.
<7> The method according to any one of <1> to <6>, wherein MMP-2 expression or TIMP-2 expression is preferably measured using protein expression, mRNA expression or promoter activation as an index.

Test Example 1 Observation of the localization of skin basement membrane constituent molecules (type IV collagen) by immunoelectron microscopy Primary antibody ( Anti-type IV collagen-mouse monoclonal IgG1, 100-fold dilution, abcam, product code: #ab6311) was reacted overnight, washed, and gold particles (diameter 1.4 nm) labeled secondary antibody (anti-mouse IgG-goat polyclonal Antibody, Nanoprobes, product code: #2001; 200-fold dilution) was allowed to react at room temperature for 2 hours. In addition, the diameter of the gold particles is increased to 30-50 nm using an amplification kit (GoldEnhance EM, Nanoprobes, product code: #2113), and the sample is fixed and dehydrated according to the standard method, then embedded in epoxy resin, sliced, and stained electronically. Electron microscopy was then performed.

The results are shown in FIG.
In FIG. 1, black dots derived from gold particles indicate portions where type IV collagen molecules are present. In the healthy subject on the left, black gold particles (one example is shown with an arrowhead) are arranged in a line on the skin basement membrane indicated by white arrows, but in the lesion of the vitiligo patient on the right, black gold particles on the skin basement membrane are observed. Gold particles were limited to a part, and most gold particles were observed in the dermis away from the skin basement membrane. From these results, it was found that the distribution of type IV collagen, which is a constituent molecule of the skin basement membrane, is uneven in vitiligo lesions.

Test Example 2 Immunostaining of MMP Frozen sections of human skin tissue from healthy subjects (5 subjects) and patients with vitiligo vulgaris (5 subjects) were reacted overnight with a primary antibody (100-fold dilution below), washed, and nuclear stained. secondary antibody (anti-mouse Alexa Fluor 555; product code: A-21424 or anti-mouse Alexa Fluor 488; product Code: A-11001 Invitrogen; 500-fold dilution) was allowed to react at room temperature for 1 hour, washed, mounted, and photographed. The fluorescence intensity per unit area was calculated from the photograph using image analysis software (Image J).
The primary antibodies used in this study are as follows: MMP-2 (anti-MMP2-mouse monoclonal IgG2a, Abcam, product code: #ab86607), MMP-9 (anti-MMP9-mouse monoclonal IgG2a, Abcam, product Code: #ab119906)

The results are shown in FIG.
The green fluorescent MMP-2 was remarkably upregulated in the dermis of vitiligo lesions compared to healthy subjects. On the other hand, it was also confirmed that the expression of MMP-9, which is fluorescently colored in red, is slightly increased in the dermis of vitiligo lesions. As a result of quantifying the fluorescence intensity from the stained images of 6 vitiligo patients and 6 healthy subjects, MMP-2 increased about 7-fold, while MMP-9 increased only about 2-fold.
In the figure, blue fluorescence indicates cell nuclei.

Test Example 3 Establishment of MMP-2 overexpressing MMP-2 overexpressing human fibroblasts in a three-dimensional skin model A vector (pEBMulti-Puro, wako) incorporating human MMP-2 (RefSeq: BC002576.2) was treated with Lipofectamine 3000 (Thermo Fisher Scientific) was introduced into human fibroblasts (lifeline; product code: FC-0001), and stable MMP-2 overexpressing human fibroblasts were established by puromycin selection.

The above MMP-2 overexpressing human fibroblasts were seeded (final concentration 2.5×10 ⁶ cells/ml) in DMEM medium supplemented with a collagen solution (5 mg/mL; Kokensha; product code: IAC-50) and transwelled. (Corning, #3412) at 37°C for 30 minutes. After culturing, the hardened collagen gel was cut in half, the half-cut gel was transferred to another transwell (Corning, #3412), and DMEM medium supplemented with collagen seeded with normal human fibroblasts was added to the empty half. The cells were injected and incubated at 37° C. to gel the collagen. After 2 days of culture, each collagen gel spontaneously integrated, with half of the collagen gel layer consisting of normal human fibroblasts (Fig. 3A, green) and the other half consisting of MMP-2-overexpressing human fibroblasts (Fig. 3A, green). A collagen gel layer containing yellow) was formed. Melanocytes (Themofisher; product code: C2025C) and keratinocytes (Kurabo; product code: KK-4009) were uniformly seeded on the gel and cultured for one week to construct a three-dimensional skin model. The culture conditions for constructing the three-dimensional skin model conformed to the conditions described in the reference (J Dermatol Sci, 2005;40:105-114). After culturing for one week, the constructed three-dimensional skin model was visually observed, and then immunostaining of laminin 5 (LN5, former name, current name: laminin 332), which is a skin basement membrane-related molecule, and TRP1, which is a melanocyte marker. We followed the steps below. A primary antibody (100-fold dilution below) was allowed to react overnight on a frozen section of a 3D skin model, and after washing, a nuclear staining agent (DAPI Solution, Wako Pure Chemical Industries; product code: 340-07971; 500-fold dilution) was applied. Mixed secondary antibodies (anti-rabbit Alexa Fluor 555 or anti-mouse Alexa Fluor 488; Invitrogen; 500-fold dilution) were reacted at room temperature for 1 hour, washed, mounted, and photographed. The primary antibodies used in this test are as follows: Laminin 5 (anti-Laminin5-mouse monoclonal IgG1, Abcam, product code: #ab78286), TRP1 (anti-TRP1-rabbit polyclonal antibody, Sigma, product code: # HPA000937)

The results are shown in FIG.
Pigmentation was observed in the left hemisphere containing normal human fibroblasts, while a depigmentation-like phenomenon was observed in the right hemisphere containing MMP-2 overexpressing human fibroblasts (Fig. 3B). . Furthermore, as a result of confirming its localization by immunostaining using TRP1, a melanocyte marker, melanocytes were present in the section of the left semicircle (Mock) containing normal human fibroblasts, which is the pigment-existing part, but detachment. No melanocytes were present in sections of the right hemisphere (pEB-MMP2) containing MMP-2 overexpressing human fibroblasts on the dye side (Fig. 3C, fluorescence in red). In addition, when immunostaining for laminin 5 (LN5), a molecule associated with the skin basement membrane, was confirmed linearly on the skin basement membrane in the Mock section where the pigment was present, pEB-MMP2 on the depigmented side was observed. In the section of , many were observed in the collagen gel away from the skin basement membrane (Fig. 3C, green fluorescence), and histological findings of type IV collagen, which is a molecule constituting the skin basement membrane in the patient with vitiligo in Test Example 1 (Fig. 1 ).
In the figure, blue fluorescence indicates cell nuclei.

Test Example 4 MMP-2 overexpression in human skin tissue culture system Human skin tissue obtained from a medical university in Japan was divided into squares of about 1 cm square, and normal human fibroblasts or MMP-2 established in Test Example 3 Overexpressed human fibroblasts (both cells pre-labeled with the fluorescent dye PKH-26) were injected into the dermis (100ul injection of ^5x106 cells/ml suspension). Thereafter, the cells were cultured in transwell for 1 week, immunostained as follows, and internal changes were observed. Primary antibody (100-fold dilution below) was reacted with frozen sections of cultured human skin tissue overnight, and after washing, secondary antibody (anti-rabbit Alexa Fluor 555 or anti-mouse Alexa Fluor 488; Invitrogen; 500-fold dilution) was allowed to react at room temperature for 1 hour, and after further washing, it was mounted and photographed. The primary antibodies used in this test are as follows: Collagen IV (anti-Collagen IV-mouse monoclonal IgG1, biogenex, product code: #AM3795M), TRP1 (anti-TRP1-rabbit polyclonal antibody, Sigma, product code: #HPA000937)

The results are shown in FIG.
The skin basement membrane (arrowhead) indicated by fluorescent staining (green) of collagen IV, which is a component of the skin basement membrane, maintained a continuous basement membrane structure in normal human fibroblast (control cell)-introduced skin. Fragmentation of the skin basement membrane was confirmed in the skin into which MMP-2 overexpressing human fibroblasts were introduced (Fig. 4, left) (Fig. 4, right). Melanocytes indicated by fluorescence staining (red) of TRP1, a melanocyte marker, were localized on the skin basement membrane in skin into which control cells were introduced (Fig. 4, left insert), whereas MMP-2 excess was observed. Loss of melanocytes (right insert in FIG. 4) was confirmed in the skin into which the expressing human fibroblasts were introduced.

Test Example 5 MMP-2 overexpression in mouse skin dermis In the same manner as in Test Example 3, a vector (pEBMulti-Puro, wako) incorporating mouse MMP-2 (RefSeq: BC070430.1) was added to Lipofectamine 3000 (Thermo Fisher Scientific ) was introduced into mouse fibroblasts (SCR; product code: M2300-57), and stable MMP-2 overexpressing mouse fibroblasts were established by puromycin selection.

KRT14-Kitl mice (Riken BRC, 9-week-old male), which have melanocytes in the epidermis as in humans, were shaved, and normal mouse fibroblasts or MMP-2-overexpressing mouse fibers were transferred to the dorsal dermis. Blast cells were injected and implanted (100 ul injection of 2.5×10 ⁶ cells/ml suspension). Three weeks after transplantation, the hair was completely removed with a hair remover, and then photographed.

The results are shown in FIG.
No obvious change in skin color was observed in the mice transplanted with normal mouse fibroblasts (FIG. 5, left). On the other hand, mice implanted with MMP-2-overexpressing mouse fibroblasts exhibited clear vitiligo (similar to human vitiligo patients) at the site of implantation (Fig. 5, right).

Claims (5)

A method for evaluating or selecting an agent for preventing or improving vitiligo, comprising the following steps (1) to (3).
(1) Step of contacting a test substance with a tissue or cell capable of expressing MMP-2 (2) Step of measuring the expression or activity of MMP-2 in the tissue or cell (3) Measured in (2) Based on the results, the step of evaluating or selecting a test substance that reduces the expression or activity of MMP-2 as an agent for preventing or improving vitiligo A method for evaluating or selecting an agent for preventing or improving vitiligo, comprising the steps of (4) to (6) below.
(4) Step of contacting a test substance with a tissue or cell capable of expressing TIMP-2 (5) Step of measuring the expression of TIMP-2 in the tissue or cell A step of evaluating or selecting a test substance that increases the expression of TIMP-2 as a vitiligo preventive or ameliorating agent based on 3. The method according to claim 1 or 2, wherein the vitiligo is vitiligo vulgaris or chemical substance-induced depigmentation. 4. The method of claim 3, wherein the chemical-induced depigmentation is rhododenol-induced depigmentation. The method according to any one of claims 1 to 4, wherein the tissue or cell capable of expressing MMP-2 or the tissue or cell capable of expressing TIMP-2 is human dermal tissue or cells derived from human dermal tissue.

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