KR20130056955A - Composition for promoting mature melanocyte migration comprising chemokine, detection kit for skin-active ingredients comprising chemokine gene and the method for detecting skin-active ingredients by using chemokine gene - Google Patents

Abstract

PURPOSE: A composition is provided to promote the migration of melanocytes in skin and hair, to relieve and treat a lesion in hypopigmentary diseases such as vitiligo and grey hair, and to quickly and simply screen the skin vitalizing substance. CONSTITUTION: A composition for promoting adult melanocyte migration contains chemokine which is selected from a group consisting of CTACK, I-TAC, Lptn, PARC, SDF1, and TARC. The composition is used for treating hypopigmentary diseases. A kit for detecting a skin vitalizing substance contains a chemokine-specific antibody, a secondary antibody, a chromogenic label, and skin cells. The kit is used for detecting the skin vitalizing substance by screening a material which regulates expression of chemokine in skin cells. A method for detecting skin vitalizing substance comprises: a step of treating the skin cells with a candidate material and identifying expression of chemokine; and a step of isolating and purifying a material which reduces expression of chemokine. [Reference numerals] (AA) Human melanocyte migration(%); (BB) Control group; (CC) Treatment material

Description

Composition for promoting melanin cell migration containing chemokine, kit for screening skin active material using chemokine gene and method for detecting skin active material using same {composition for promoting mature melanocyte migration comprising chemokine, detection kit for skin-active ingredients comprising chemokine gene and the method for detecting skin-active ingredients by using chemokine gene}

The present invention relates to a composition containing chemokine to promote adult melanin cell migration in the skin or hair, and more particularly to a composition containing chemokine gene to promote adult melanin cell migration for use in treating hypopigmentary diseases. The hypopigmentation disease is characterized by vitiligo or white hair.

In addition, the present invention relates to a kit for screening the skin active material and a method for searching for skin active material using the same, and more particularly, to induce chemokine gene expression in skin cells by using a kit including a chemokine (chemokine) gene The present invention relates to a skin active substance searching kit for searching for skin active substances such as a whitening substance and a substance for treating hypopigmentary diseases by separating and purifying candidate substances, and a method for searching for skin active substances using the same.

Cell migration is one of normal cell physiological activities and occurs in tissue development and differentiation, migration due to activation of immune cells, formation of new blood vessels, and regeneration of damaged tissue. Inadequately regulated cell migration can lead to development-related diseases such as cerebral nervous system disease caused by abnormal neural network formation, inflammatory diseases caused by abnormal immune cell activation, such as abnormal rheumatoid arthritis, and cell migration to repair damaged tissues. Various diseases may appear, such as delayed wound healing, metastasis of cancer, etc.

Melanocytes develop from neural crest cells, which are precursors, and develop into melanocytes. Melanocyte progenitor cells differentiate into adult melanocytes and are finally distributed to the basal epidermis, hair follicle, and iris of the skin epidermis to maintain a constant number. The migration of melanocyte progenitor cells is well known, and various signaling mechanisms and transcription factors have been identified. However, little has been studied about the migration of adult melanocytes. Recently, however, the necessity of research on the movement of melanocytes, such as the study of abnormal activation of melanocytes in the skin by ultraviolet rays, as well as diseases such as leukemia and vitiligo, has emerged.

Inflammation such as acne, various types of immune cells are transferred to the affected area by chemokines secreted by macrophages. In addition, recently, exposure of UVB or the like has been abnormally promoted the movement of mouse melanocytes, melanocytes limited to the hair follicles has been reported in the literature to move to the epidermis and distribution.

Blemishes and surpluses are representative pigmentation disorders, and the collapse of the basement membrane between the epidermis and the dermis has been reported. Due to the collapse of the basement membrane, melanocytes are likely to migrate to the dermis and cause pigmentation diseases such as blemishes and surpluses. Therefore, by inhibiting the migration of melanocytes to the dermis due to the collapse of the basement membrane, refractory pigmentation diseases such as blemishes can be prevented.

Vitiligo is a disease characterized by low pigments in the skin and hair. It occurs in all races and occurs in 0.5% of the world. There is no clear pathogenesis for the reduction of the number of melanocytes. Thus, no definitive treatment for vitiligo has been developed. Topical corticosteroid application, phototherapy, photochemotherapy, and the like have been used to enhance pigmentation in vitiligo patients. The treatment using such UV rays aims to induce the proliferation of melanocytes and the migration of melanocytes to the affected areas. Experimental results have reported that melanocyte progenitor cells in the outer root sheath of hair follicles are proliferated by UV rays, and the increased melanocyte progenitor cells migrate to the surrounding skin so that the skin tone of vitiligo lesions is similar to that of normal areas. . Therefore, the chemokines of the present invention that induce the migration of adult melanocytes can be used for hypopigmentation diseases such as vitiligo and white hair.

The skin is a finely regulated immune organ that secretes several cytokines, chemokines, and other inflammatory mediators. Chemokines are immunoreactive mediators with a small size of about 8-14 kD and exhibit selective chemotaxis to surrounding cells, inducing cell migration and integration into target organs. It is well known that cytokines already play an important role in skin physiology such as atopy and contact dermatitis, while chemokines are not well known in skin physiology.

The present inventors have found that certain chemokines affect the migration of adult melanocytes, and found that chemokines can be used to promote melanocyte migration of skin or hair, thereby completing the present invention. It is therefore an object of the present invention to provide a composition containing chemokines which promotes adult melanocyte migration in the skin or hair.

In addition, the present inventors have made a kit containing chemokine to search for skin active substances such as whitening substances and hypopigmental disease treatment substances by confirming whether the expression of chemokine in the skin cells such as keratinocytes, fibroblasts or melanocytes. It has been found that the present invention has been completed. Therefore, another object of the present invention is to provide a search kit for screening the skin active material through the presence of chemokine gene expression in skin cells and a method for searching the skin active material using the same.

In order to achieve the above object, the present invention provides a composition containing chemokine to promote adult melanocyte migration. More specifically, the composition containing chemokine to promote adult melanocyte migration in the skin or hair, wherein the chemokine promotes the migration of adult melanocytes, preferably CTACK, I-TAC, Lptn, PARC, SDF1, TARC , RANTES, IP10, and at least one member selected from the group consisting of Eotaxin. The composition can be used for the treatment of diseases characterized by low pigmentation of the skin, such as vitiligo, white hair.

In addition, the present invention relates to a method for searching for the amount of chemokine expression in skin cells, and through this screening for substances that can control the expression of chemokine in the skin cells it can search for skin active substances. The chemokine is to promote the migration of adult melanocytes, preferably characterized in that at least one selected from the group consisting of CTACK, I-TAC, Lptn, PARC, SDF1, TARC, RANTES, IP10, and Eotaxin. More preferably, one or more selected from the group consisting of CTACK, I-TAC, Lptn, PARC, SDF1, and TARC can be used.

More specifically, the present invention relates to a method of searching for a whitening substance by screening a substance that reduces the expression of chemokines in the skin cells. The whitening material may include all of the whitening material that improves pigmentation after inflammation, such as acne, acne, whitening material that brightens skin tone or improves skin pigmentation such as blemishes and surpluses by reducing melanocyte migration.

The present invention also relates to a method for searching for a substance for treating hypopigmentary disease by screening a substance for promoting chemokine expression in the skin cells. The hypopigmentation disease refers to a disease exhibiting hypopigmentation in skin, hair, and the like, preferably vitiligo, white hair, and the like.

The search kit of the present invention which can detect the expression level of chemokines in skin cells is composed of chemokine specific antibodies, secondary antibodies, coloring labels and skin cells, and controls the expression level of chemokines in the skin cells. Screening of the substance can search for skin active substances. The chemokine is one or more selected from the group consisting of CTACK, I-TAC, Lptn, PARC, SDF1, TARC, RANTES, IP10, and Eotaxin as promoting the migration of adult melanocytes.

The chemokine specific antibody may be used as a capture antibody contained in commercial kits for CTACK, I-TAC, Lptn, PARC, SDF1, TARC, RANTES, IP10, and Eotaxin, but is not limited thereto. Also preferably, CTACK capture antibody (MAB3761, R & D systems), I-TAC capture antibody (MAB672, R & D systems), Lptn capture antibody (MAB6951, R & D systems), PARC capture antibody (MAB394, R & D systems), SDF1 capture antibody ( AF-351-NA, R & D systems), TARC capture antibody (MAB364, R & D systems), RANTES capture antibody (MAB678, R & D systems), IP10 capture antibody (MAB266, R & D systems), Eotaxin capture antibody (MAB320, R & D systems) Can be used.

The secondary antibody may be CTACK, I-TAC, Lptn, PARC, SDF1, TARC, RANTES, IP10, and Eotaxin specific detection antibody (Detection antibody), but is not limited thereto. Also preferably, CTACK detection antibody (BAF376, R & D systems), I-TAC detection antibody (BAF672, R & D systems), Lptn detection antibody (BAF695, R & D systems), PARC detection antibody (BAF394 R & D systems), SDF1 detection antibody (BAF351 , R & D systems), TARC detection antibody (BAF364, R & D systems), RANTES detection antibody (BAF278, R & D systems), IP10 detection antibody (BAF266, R & D systems), Eotaxin detection antibody (BAF320, R & D systems).

As the label for color development, streptavidin-HRP (Streptavidin-HRP) contained in commercial kits for CTACK, I-TAC, Lptn, PARC, SDF1, TARC, RANTES, IP10, and Eotaxin may be used. It is not limited. It is also preferable to use streptavidin conjugated to horseradish-peroxidase to which horseradish peroxidase is bound.

More specifically, the present invention provides a search kit for searching for a whitening material by screening a substance for reducing chemokine expression in the skin cells. The whitening material may include all of the whitening material that improves pigmentation after inflammation, such as acne, acne, whitening material that brightens skin tone or improves skin pigmentation such as blemishes and surpluses by reducing melanocyte migration.

In another aspect, the present invention provides a search kit for searching for a substance for treating hypopigmental disease by screening a substance for promoting chemokine expression in the skin cells. The hypopigmentation disease refers to a disease exhibiting hypopigmentation in skin, hair, and the like, preferably vitiligo, white hair, and the like.

In one embodiment of the present invention, the skin cells are normal human epidermal keratinocytes (NHEK), fibroblasts (NHF, normal human fibroblasts), melanocytes (NHEM, normal human epidermal melanocyte), human epidermal-derived immune cells And it may be one or more selected from cells co-cultured with human keratinocytes and melanocytes, but is not limited thereto.

In another aspect, the present invention using the search kit, the step of confirming the expression of chemokine after treating the candidate material to the skin cells; And it relates to a method for searching the skin active material through the step of separating and purifying the substance that reduces chemokine expression.

The composition according to the present invention can be applied to the affected area in the hypopigmentation disease such as vitiligo and white hair by promoting the migration of melanocytes in the skin and hair, thereby exhibiting a symptomatic relief and therapeutic effect.

In addition, the skin active substance search kit according to the present invention can quickly and easily screen skin active substances such as whitening substances and hypopigmentary disease treatment substances through the expression of chemokine gene in skin cells.

1 is a graph showing the results of confirming the cell migration in chemokine-treated human melanin cells.
Figure 2 is a graph showing the results of confirming the cell migration in chemokine-treated human keratinocytes.
3 is a graph showing the results of confirming the expression level of RANTES in human keratinocytes and human melanocytes treated with kojic acid.
4 is a graph showing the results of confirming the amount of RANTES expression in human epidermal-derived immune cells treated with ginsenoside F1.

Hereinafter, the present invention will be described more specifically with reference to examples and test examples. These examples are provided only for understanding the contents of the present invention, but the scope of the present invention is not limited to these examples and test examples, and modifications, substitutions, and insertions commonly known in the art may be performed. It may be included in the scope of the present invention.

 [ Reference Example  1] Preparation of melanocytes

Normal human epidermal melanocyte-moderately pigmented (C-102-5C) purchased from Cascade was planted in 100 x 2 plates in 6 x 10 ⁵ counts, and the basal medium was HMGS (Cat. No. S-002-5, 254 medium (product number M-254-500, Gibco) containing Gibco) was used and incubated in a 37 ° C., 5% CO ₂ incubator. Subcultures were obtained at a density of about 70-80%, and melanocytes were provided to subsequent experiments.

[ Reference Example  2] Preparation of keratinocytes

 Normal human epidermal Keratinocytes were used NHEK-Neo, Pooled (Neonatal Normal Human Epidermal Keratinocytes, Pooled, Article No. 00192906, Lonza) provided by Lonza. KGM-Gold BulletKit (Product No. 192060, Lonza) can be used as the basal medium. The dish was coated with 0.1% gelatin to maintain the propagation property of keratinocytes on the basement membrane.

Human keratinocytes were planted in 6 × 10 ⁵ cells in 100 cm 2 plates, and cultured in 37 ° C., 5% CO ₂ incubator in basal medium. Subcultures were made at a density of about 70-80%, and keratinocytes were provided to subsequent experiments.

[ Reference Example  3] Preparation of skin-derived immune cells

CD3 ⁺ epidermal T cells (CD3 ⁺ epidermal T cells) are obtained from human skin tissue. The skin tissue is separated from the dermis and the epidermis by basal medium (2 mM L-glutamine, 100 U / ml penicillin, 100) containing 2 units / ml of Dispase II solution (product number 4942078001, Roche). Incubate for 12 hours at 4 ° C in RPMI 1640 medium (Product No. 12-115F, Lonza) containing mg / ml streptomycin and inactivated 10% fetal calf serum. After separating the epidermal layer using sterile forceps, 2 unit / ml dispase II solution was added to the epidermal layer and incubated at room temperature for 10 minutes. 10 mM EDTA (product number E6758, Sigma) is added to inactivate the enzyme. A suspension (cell strainer 100 mm; product number REF352360, BD falcon) is obtained to obtain a suspension containing lymphocytes. Cells were separated using a centrifuge (Fincoll-Paque gradient centrifugation, Hitopagne-1077, product number 1077-1, Sigma), and then 50 units / ml of interleukin 2 (IL-2, product number 202-IL, R & D systems). ) Was incubated for 24 hours at 37 ℃ in the basal medium containing. To isolate only T cells, the cultured cells and basal medium were placed in a 100 mm culture dish coated with a CD3 antibody (anti-CD3 antibody, product number MBA100, R & D systems), and cultured for 6 days to obtain T cells.

 [ Reference Example  4] of human keratinocytes and human melanocytes Co-culture

Human melanocytes (Normal Human Epidermal Melanocyte-Moderately pigmented, product number C-102-5C, Cascade) and the human keratinocytes (Neonatal Normal Human Epidermal Keratinocytes, Pooled, product number 00192906, Lonza) in a ratio of 1: 5 Specifically, KGM-Gold BulletKit (product number: 1,25 x 10 ⁵ human keratinocytes cultured in Reference Examples 1 and 2 and 2.5 x 10 ⁴ human melanocytes were mixed in a 6 well plate. In a medium containing 1: 1 mixture of 254 medium (model number M-254-500, Gibco), including 192060, Lonza) and HMGS (model number S-002-5, Gibco), in a 37 ° C, 5% CO ₂ incubator. Incubate for 24 hours before use in experiments.

[ Test Example  One] On chemokines  Migration of melanocytes

In order to determine whether the movement of melanocytes is induced by chemokines, human melanocytes were treated with chemokines secreted from immune cells as well as from skin cells, and FK506 (Sigma Aldrich, a product reported by melanocyte migration) has been reported. Number F4679) was used as a control. Chemokines were purchased from the R & D system, and the product numbers were CTACK (product number 376-CT), Fracktalkin (product number 362-CX), MCP1 (product number 279-MC), MCP4 (product number 327-P4), and MIP1b ( Article number 271-BME), PARC (model number 394-PA), RANTES (model number 278-RN), IP10 (model number 266-IP), Eotaxin (model number 320-EO), and I-TAC (model number 672 -IT), MIG (model number 392-MG), MIP1a (model number 270-LD), SDF1 (model number 350-NS), TARC (model number 364-DN), Lptn (model number 695-LT), MCP2 (Product number 281-CP) and MDC (product number 336-MD) were used.

Human melanocytes cultured in Reference Example 1 were incubated in a basic medium (254medium, product number M-254-500; Gibco) without HMGS at 37 ° C., 5% CO ₂ incubator for 4 hours. The cultured human melanin cells were separated using trypsin / EDTA, incubated for 30 minutes at 37 ° C. in a basal medium containing 0.1% BSA, and lightly tapped the tube containing the cells at 10 minute intervals.

200μg / ml of chemokine CTACK, Fracktalkin, MCP1, MCP4, MIP1b, PARC, RANTES, IP10, Eotaxin, I-TAC, MIG, MIP1a, SDF1, TARC, Lptn, MCP2, MDC in a basic medium containing 0.1% BSA After diluting with and preparing 27 μl in the lower chamber of the Boyden chamber, melanin cells treated with nothing as a control were treated with 1 μM of FK506 as a positive control.

The gasket and the upper chamber are assembled in order, with the glossy side of the membrane facing down on the lower chamber. After dispensing the cultured human melanin cells in the upper chamber by 104 cells / 56 μl, the assembled Boyden chamber is incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator. Stain the membranes using H & E staining (Hematoxylin and Eosin staining), place them on a glass plate with the shiny side in contact with the glass, and wipe the unmoved cells with a cotton swab. Figure 6 shows the results of calculating the movement of melanocytes by counting the number of cells moved by taking 6field pictures for each well by Equation 1 below.

As shown in Figure 1 of the chemokines used in the experiments CTACK, I-TAC, Lptn, PARC, SDF1, TARC, RANTES, IP10 and Eotaxin induced the migration of melanocytes more than 1.5 times compared to the control. However, eight other chemokines did not induce melanocyte migration. From this, it can be seen that not all chemokines secreted from the skin nonspecifically move melanocytes but only specific chemokines induce melanocyte migration.

[ Test Example  2] On chemokines  Migration of keratinocytes by

In order to determine whether the chemokine of the present invention induces the migration of skin cells other than melanocytes, the chemokine migration of keratinocytes, which are the major constituent cells of the skin epidermis in which melanocytes exist, was examined.

Chemokine CTACK, I-TAC, Lptn, PARC, SDF1, TARC, which induced the migration of melanocytes in Test Example 1, were used, and the control group used keratinocytes without any treatment.

Human keratinocytes cultured in Reference Example 2 were extracted from bovine pituitary extract (BPE), human epidermal growth factor (hEGF), bovine insulin, hydrocortisone (Hydrocortisone), 37 ° C., CO ₂ in a base medium (KGM-Gold ™ BulletKit ™, product no. 192060, Lonza) containing epinephrine and transferrin Incubated for 4 hours in an incubator. The cultured human keratinocytes were separated using trypsin / EDTA and incubated for 30 minutes at 37 ° C. in a basic medium containing 0.1% BSA (KGM-Gold ™ BulletKit ™, product no. 192060, Lonza) at 10 minute intervals. Tap the tube containing the.

The chemokine CTACK, I-TAC, Lptn, PARC, SDF1, TARC was prepared by diluting 200 μg / ml each in a basic medium containing 0.1% BSA (KGM-Gold ™ BulletKit ™, product no. 27 μl was dispensed into the lower chamber of the Boyden chamber, and keratinocytes treated with nothing were used as a control.

The gasket and the upper chamber are assembled in order, with the glossy side of the membrane facing down on the lower chamber. After dispensing the cultured human keratinocytes into the upper chamber by 104 cells / 56 μl, the assembled Boyden chamber is incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator. Stain the membranes using H & E staining (Hematoxylin and Eosin staining), place them on a glass plate with the shiny side in contact with the glass, and wipe the unmoved cells with a cotton swab. Figure 6 shows the results of calculating the movement of keratinocytes by Equation 1 by counting the number of cells moved by taking 6 fields for each well.

As shown in Figure 2 chemokine CTACK, I-TAC, Lptn, PARC, SDF1, TARC induced melanocyte migration, but did not induce the migration of keratinocytes. Therefore, it can be seen that the increase in secretion of the chemokine in the skin by a specific stimulus does not induce the migration of keratinocytes and the melanocytes are specifically migrated.

[ Example  One] Chemokine  Screening of Skin Active Substances (Kojic Acid) by Expression Change

1.25 x 10 ⁵ human keratinocytes and 2.5 x 10 ⁴ human melanocytes co-cultured in Reference Example 4 were subjected to KGM-Gold BulletKit (product no. 192060, Lonza) and HMGS (product no. S-002-5, Gibco). ) Was treated with 200 mM of known kojic acid (Kojic acid, product number K3125, Sigma Aldrich) in a test medium containing 1: 1 mixture of 254 medium (product number M-254-500, Gibco) at 37 ° C. Incubated for 48 hours in a 5% CO ₂ incubator to obtain a supernatant. As a control, the supernatant was obtained by incubating the co-cultured cells for 48 hours in the same manner, except that the co-cultured cells were not treated with kojic acid.

100 ml of the supernatant obtained above was put into each well of a 96 well plate, and 100 μl of a specific antibody (MAB678, R & D systems) to RANTES diluted to 1 μg / ml with PBS was added at 4 ° C. Incubated for 12 hours. In order to obtain a standard curve, the protein concentration of recombinant human RANTES, a reference material, was serially diluted to 300, 150, 75, 37.5, 18.75, 9.4, 4.7 ng / ml. After washing three times with washing buffer (PBS containing 0.05% Tween 20), and adding a blocking solution containing 1% skim milk (PBS containing 10% FBS) to prevent nonspecific reactions Incubated at room temperature for 1 hour. After removing the antigen-containing PBS, and washed three times with a buffer solution, the remaining solution was completely removed using a paper towel. After washing, 100 μl of the sample was added and reacted at room temperature for 2 hours to attach the antigen to the plate to which the antibody was attached. After removing the samples, each well was washed three times with a wash buffer solution. 100 μl of a detection antibody (Biotinylated anti-human RANTES) diluted to 20 ng / ml was added and incubated at room temperature for 2 hours. After washing twice with washing buffer, and added streptavidin conjugated to horseradish-peroxidase diluted 1: 200 for color reaction, incubated for 20 minutes at room temperature do. The wash is washed three times with a cleaning buffer solution. At this time, remove the solution completely by tapping it gently with a paper towel so that there is no remaining solution. Hydrogen peroxide solution and tetramethylbenzidine were mixed 1: 1 to put 100 μl into each well to induce a color reaction and incubate at room temperature for 20 minutes. 50 μl of 2N sulfuric acid was added to terminate the color reaction, and the absorbance was measured at 450 nm using a spectrophotometer. The results are shown in FIG. 3.

As shown in the results of FIG. 3, when kojic acid was treated to cells co-cultured with human keratinocytes and human melanocytes, it was confirmed that the expression of RANTES was reduced compared to the control group, which was not treated by the present invention. It can be seen that the whitening material can be explored.

[ Example  2] Chemokine  Skin active substance by expression change Gin Senocide  F1)

The CD3 ⁺ epidermal-derived culture in Reference Example 3 T cell (CD3 ⁺ epidermal T cell) a CD3 antibody (immobilized anti-CD3 antibody) was fixed on the plate to activate, 200 U / ml interleukin-2 (IL-2) 500ng / ml PHA is added to the basal medium and treated with 100 mM Ginsoeniside F1, a known whitening substance. After culturing for 48 hours in a 5% CO ₂ incubator at 37 ° C, a supernatant was obtained. Controls were immobilized anti CD3 antibody, 200 U / ml interleukin 2 (IL-2) for activation. T cells were added to 500 ng / ml PHA (Phytohaemaglutinin; product # L1668; Sigma) but without Ginsoeniside F1.

100 ml of the supernatant obtained above was put into each well of a 96 well plate, and 100 μl of a specific antibody (MAB678, R & D systems) to RANTES diluted to 1 mg / ml with PBS was added at 4 ° C. Incubated for 12 hours. In order to obtain a standard curve, the protein concentration of recombinant human RANTES, a reference material, was serially diluted to 300, 150, 75, 37.5, 18.75, 9.4, 4.7 ng / ml. After washing three times with washing buffer (PBS containing 0.05% Tween 20), and adding a blocking solution containing 1% skim milk (PBS containing 10% FBS) to prevent nonspecific reactions Incubated at room temperature for 1 hour. After removing the antigen-containing PBS, and washed three times with a buffer solution, the remaining solution was completely removed using a paper towel. After washing, 100 μl of the sample was added and reacted at room temperature for 2 hours to attach the antigen to the plate to which the antibody was attached. After removing the samples, each well was washed three times with a wash buffer solution. 100 μl of a detection antibody (Biotinylated anti-human RANTES) diluted to 20 ng / ml was added and incubated at room temperature for 2 hours. After washing twice with washing buffer, and added streptavidin conjugated to horseradish-peroxidase diluted 1: 200 for color reaction, incubated for 20 minutes at room temperature do. The wash is washed three times with a cleaning buffer solution. At this time, remove the solution completely by tapping it gently with a paper towel so that there is no remaining solution. Hydrogen peroxide solution and tetramethylbenzidine were mixed 1: 1 to put 100 μl into each well to induce a color reaction and incubate at room temperature for 20 minutes. 50 μl of 2N sulfuric acid was added to terminate the color reaction, and the absorbance was measured at 450 nm using a spectrophotometer. The results are shown in FIG. 4.

As shown in the results of Figure 4, when treated with human epidermal-derived immune cells Ginsenoside F1, it can be seen that the expression of RANTES is reduced compared to the untreated group, the whitening material can be searched by the search kit of the present invention It can be seen that.

Claims (14)

A composition for promoting adult melanin cell migration containing chemokines. The composition of claim 1, wherein the chemokine is at least one selected from the group consisting of CTACK, I-TAC, Lptn, PARC, SDF1, and TARC. The composition of claim 1 or 2, wherein the composition is used for treating hypopigmentary disease. The composition for promoting adult melanocyte migration according to claim 3, wherein the hypopigmentation disease is vitiligo. 4. The composition of claim 3, wherein the hypopigmentation disease is white hair. Consisting of a chemokine specific antibody, a secondary antibody, a coloring label and skin cells,
A search kit for searching for skin active substances by screening for substances regulating chemokine expression in the skin cells. 7. The search kit of claim 6, wherein the chemokine promotes adult melanocyte migration. The search kit of claim 6, wherein the chemokine is at least one selected from the group consisting of CTACK, I-TAC, Lptn, PARC, SDF1, TARC, RANTES, IP10, and Eotaxin. The method of claim 6, wherein the skin cells are selected from the group consisting of human keratinocytes, fibroblasts, melanocytes, human epidermal-derived immune cells and cells co-cultured with human keratinocytes and melanocytes. Navigation kit. 10. The kit according to any one of claims 6 to 9, wherein the substance regulating chemokine expression reduces chemokine expression. 11. The kit of claim 10 wherein the skin active material is a whitening material. 10. The kit according to any one of claims 6 to 9, wherein the substance regulating chemokine expression increases chemokine expression. 13. The kit according to claim 12, wherein the skin active substance is a substance for treating hypopigmentary disease. Using the search kit according to any one of claims 6 to 9,
Confirming the expression of chemokines after treating candidate skin cells; And
Isolating and purifying substances that reduce chemokine expression; Searching for a skin active material comprising a.

Images