WO2013058578A2

WO2013058578A2 - System for screening skin-whitening material

Info

Publication number: WO2013058578A2
Application number: PCT/KR2012/008555
Authority: WO
Inventors: 이애영; 이태룡; 최현정
Original assignee: (주)아모레퍼시픽; 동국대학교 산학협력단
Priority date: 2011-10-18
Filing date: 2012-10-18
Publication date: 2013-04-25
Also published as: WO2013058578A3

Abstract

Disclosed are a system for screening a skin-whitening material comprising one or more layers which include one or more cells of fibroblasts, keratinocytes, and melanocytes, and a method for screening a skin-whitening material using the system. In addition, the invention presents a composition for skin whitening which comprises a material screened by the method for screening a skin- whitening material as an active ingredient.

Description

【Specification】

[Name of invention]

Skin Whitening Substance Screening System

Technology Field

The present invention relates to a skin lightening material screening system and a method of screening skin lightening material.

Background Art

<2> In developing new whitening substances, candidate substances are directly treated with melanocytes (melanocytes), which are melanocyte-producing cells, to determine whether they inhibit melanin production, or irradiation of melanocytes with ultraviolet rays and candidate substances occur at this time. It has been used to determine whether or not to block melanin-producing signals. However, in real skin, melanocytes do not exist alone and produce melanin under physical or chemical effects directly or indirectly from other surrounding cells or microenvironments. Melanosite alone reproduces this state of skin tissue. Can not.

<3> The hyperpigmentation of the skin, such as blemishes, spots, and black spots, differs in the causes and mechanisms of the increase in melanin production by UV rays. In the absence of this, melanin production and dispersion continue to increase. Therefore, in the case of the whitening material developed based on the existing UV irradiation method, in many cases, the clinical trial does not show the effect of inhibiting human skin hyperpigmentation. In addition, biopsy of the skin of a subject's hyperpigmentation site to study skin hyperpigmentation is not only costly and laborious, and difficult to obtain human skin tissue. Animal models of microdepressants are also difficult to obtain.

Therefore, there is a need for a system and a screening method for screening a whitening substance which are very similar to the structure of human skin tissues, particularly human skin tissues exhibiting skin hyperpigmentation.

<5>

<6> [Preceding Technical Documents]

<7> [Patent Documents]

<8> Korean Patent Registration No. 10-0840144 (registered on June 16, 2008)

[Detailed Description of the Invention]

[Technical problem] An aspect of the present invention is to provide a skin whitening material screening system capable of screening an effective and simple clinically useful skin whitening material, and a method for screening a skin whitening material using the same.

Another aspect of the present invention is to provide a skin whitening composition that exhibits an excellent skin whitening effect, including the material screened according to the skin whitening material screening method as an active ingredient.

In addition, another aspect of the present invention is to provide a composition for skin whitening or external composition for skin comprising the melanogenesis gene involved in the skin itself as an active ingredient.

Technical Solution

One aspect of the present invention provides a skin whitening material screening system comprising one or more layers comprising one or more cells of fibroblast, keratinocyte and melanocyte hyperplasia.

Another aspect of the present invention provides a method for screening a skin whitening substance treated with a candidate substance, wherein at least one cell of fibroblasts, keratinocytes and melanocytes is used.

It provides a skin whitening material screening method comprising the step of determining the degree of expression or phosphorylation of one or more of WIF 1, WNT 1, WNT 5a and NFATC 2.

Another aspect of the present invention provides a skin whitening composition comprising the material screened according to the skin whitening material screening method as an active ingredient or a skin whitening composition and a skin external composition comprising WIF 1 itself as an active ingredient. To provide.

Advantageous Effects

Skin whitening material screening system according to the present invention is designed to be close to the actual skin tissue can be easily screened clinically whitening material in a small time and cost in vitro. In particular, unlike conventional methods of screening whitening substances by irradiating ultraviolet rays after treating candidate melanocytes with a candidate substance, the skin whitening substance screening system according to the present invention has a gene expression level of actual skin tissue showing hyperpigmentation and Since the conditions are similar to the phosphorylation degree of the signal transduction factor, it can be easily screened for skin whitening substances, especially those effective for improving hyperpigmentation. This may be a useful system or kit for the development of new skin whitening substances, specifically hyperpigmentation improving substances, in that it is difficult to obtain human and animal models for hyperpigmentation studies.

[Brief Description of Drawings] ^

<i 6> FIG. 1 is a graph comparing differences in the expression level of WIF 1 gene between normal skin (N) and hyperpigmentation site (L) of a subject.

FIG. 2 shows WNT 1 and TOT of normal skin (N) and hyperpigmentation site (L) of a subject.

It is a graph comparing the difference in the degree of expression of 5a.

3 shows WIF in melanocytes (MC), keratinocytes (KC) and fibroblasts (FB).

1 is a graph comparing the difference in gene expression.

Figure 4 is a result showing the increase in the expression of tyrosinase (tyrosinase) in the whitening screening system including fibroblasts that inhibited WIF 1 expression.

5 is a result showing that the movement of melanosomes increased in the whitening screening system including fibroblasts that inhibited WIF 1 expression.

6 is a result showing that NFATC 2 is dephosphorylated in a whitening screening system including fibroblasts that inhibited WIF 1 expression.

FIG. 7 is an experimental result showing an increase in the expression of tyrosinase in a whitening screening system containing keratinocytes that inhibited WIF 1 expression.

FIG. 8 shows the increased melanosomal migration in the whitening screening system containing keratinocytes that inhibited WIF 1 expression.

FIG. 9 shows the results of dephosphorylation of NFATC 2 in a whitening screening system containing keratinocytes that inhibited WIF 1 expression.

FIG. 10 is a result showing that WIF 1 expression of melanocytes is increased when human WIF 1 prepared by genetic recombination technology is treated with melanocytes.

11 is a result showing that the expression of tyrosinase in the melanocytes induced the expression of WIF 1 as shown in FIG.

<2?> Figure 12 is a result showing that the movement of the melanosomes is reduced when the human WIF 1 made by genetic recombination technology in a whitening screening system including melanocytes.

FIG. 13 is a result showing that NFATC 2 is phosphorylated in melanocytes induced with expression of WIF 1 as shown in FIG. 10.

[Best Mode for Implementation of the Invention]

As used herein, the term "skin" refers to a tissue covering the body surface of an animal, and is a broad concept including not only tissues covering the body surface such as the face or body, but also the scalp and hair. <3i> Melanocytes in the skin are cells that make melanin, which determines the color of the skin, and are located on the basement membrane, which serves as the boundary between the dermis and the epidermis of the skin. Developmentally, melanocytes depart from neural-crest eel is and finally move through the melanocyte stem cell and the progenitor melanoblast (1 ^ 131101) 1 &51; It is known to differentiate into melanocytes, which are pigment cells capable of producing melanin.

The differentiated melanocytes are not present in the skin, but are produced by melanin binding to keratinocytes, keratinocytes, and fibroblasts in the dermis. Melanin from melanocytes travels to the surrounding keratinocytes through dendrite and is widely dispersed on the skin surface, protecting the skin from harmful factors such as ultraviolet rays and displaying skin color. Normal skin produces more melanin and increases melanin dispersal than usual when irritation such as ultraviolet rays occurs, then returns melanin production and dispersal back to low levels when the irritation disappears.

As discussed above, whitening has been developed by conventional methods that melanocytes do not reflect the actual skin conditions that produce melanin under complex influences from physical and chemical factors of other cells in the skin or the surrounding microenvironment. Substances, especially whitening materials developed by UV irradiation methods, often have a clinically low effect in improving skin hyperpigmentation. Since hyperpigmentation is a skin degradation caused by various causes and specific mechanisms, it is necessary to make an experimental model similar to the real skin and develop a whitening substance using it in order to develop a whitening substance that can effectively improve it. .

<34>

Hereinafter, the present invention will be described in detail.

One aspect of the invention provides a skin whitening material screening system comprising one or more layers comprising one or more cells of fibroblasts, keratinocytes and melanocytes. Another aspect of the invention is a fibroblast layer comprising a fibroblast; Provided is a skin lightening material screening system comprising a keratinocyte layer comprising keratinocytes and a melanocyte layer comprising melanocytes. At this time, the fibroblast layer is stacked on the bottom, the melanocyte layer is stacked on top of it, and the keratinocyte layer may be stacked on top of it, so as to resemble the actual skin condition. When screening the skin whitening material according to another aspect of the present invention The stem is a fibroblast layer comprising fibroblasts; And keratinocytes and melanocytes in the same layer, including layers comprising one or more of keratinocytes and melanocytes. At this time, the fibroblast layer may be stacked below, and a layer containing melanocytes and keratinocytes may be stacked thereon. The skin whitening material screening system is a three-dimensional three-dimensional structure of the skin cell layer is stacked, including not only melanocytes but also surrounding cells, it can reflect the actual skin condition well. The skin whitening material screening system according to another aspect of the present invention may include a layer including melanocytes and keratinocytes, which are known to play a more important role in melanogenesis, except for the fibroblast layer.

The fibroblast layer of the skin whitening material screening system according to one aspect of the present invention reproduces the dermal layer and includes a layer containing fibroblasts in collagen. Specifically, the fibroblasts may be a layer prepared by putting fibroblasts in collagen solution and hardening. In another aspect of the invention, the collagen is 0.001 to 30% by weight, specifically 0.01 to 20% by weight, more specifically 0.1 to ¾ to 10% by weight based on the total weight of the skin lightening material screening system May be included. When included in the above range it is appropriate to show the intended effect of the present invention, it may be appropriate to use the above range in terms of cost-effectiveness.

In the skin lightening material screening system according to an aspect of the present invention, one or more cells of fibroblasts, keratinocytes and melanocytes are selected from one or more selected from birds and pigs such as humans, mice, guinea pigs, and chickens. Contains derived cells. Skin whitening material screening system according to an aspect of the present invention is not only a material that has a skin whitening effect, in particular, a material that improves skin damage caused by ultraviolet rays, in particular, blemishes, spots, gums or gums Effective screening for substances that improve skin hyperpigmentation, such as mushrooms.

In the skin showing hyperpigmentation, the present inventors have found that WIF Kffnt inhibitory factor 1, ACCESSION NP_009122, VERSION NP_009122.2 GI: 111125011), which is an extracellular signal molecule associated with embryonic growth regulation, is reduced. Specifically, the expression of WIF 1 gene was reduced in keratinocytes and fibroblasts, not melanocytes, which are melanocytes, but also affected melanogenesis in melanocytes. In skin with decreased WIF 1 gene expression, melanin diffusion into keratinocytes was increased with the expression of tyrosinase involved in melanogenesis. This increase was found to have a close relationship between WIF 1 and skin hyperpigmentation. The expression of the WIF 1 gene is involved in the expression of GSK-3P (Glycogen synthase kinase-3 | 3) and β-catenin (β-catenin) phosphorylation 'MITFGnicrophthalmi a—associated transcription factor, which affects skin hyperpigmentation. It also confirmed that.

Accordingly, an aspect of the present invention provides a skin whitening screening system in which WIF 1 expression of fibroblasts, keratinocytes and melanocytes is suppressed, specifically, WIF 1 expression of keratinocytes or fibroblasts. Provided is a suppressed skin lightening material screening system. In another aspect of the present invention, the skin lightening material screening system includes cells in which WIF 1 expression is suppressed using siRNA. The screening system reflects hyperpigmentation skin condition with reduced WIF 1 expression of keratinocytes or fibroblasts, and can almost reproduce the skin condition showing hyperpigmentation. Therefore, in the case of using the screening system, it is possible to effectively select a material that is effective in improving skin whitening, particularly a skin pigmentation effect.

In addition, the present inventors have found that the proto-oncogene precursor WNT KWingless-type MMTV integration site family, member 1, ACCESSION NP_005421, VERSION NP_005421.1 GI: 4885655) and protein WNT 5a in skin with hyperpigmentation (Wingl ess-type MMTV integration site family, member 5a, ACCESSION NP_001243034, VERSION NP_001243034.1 GI: 371502087) Increased gene expression, NFATC2 (nuclear factor of activated T cells, cytoplasmic, calcineur in-dependent 2) ) Is dephosphorylated, and in one aspect of the present invention, one or more cells of islet infant cells, keratinocytes and melanocytes are cells in which WNT 1 and WNT 5a expression is increased or NFATC 2 is dephosphorylated. Provides a whitening material screening system. Since the screening system can reproduce the skin condition showing the hyperpigmentation layer close to reality, it is possible to effectively select a substance that is effective in improving skin hyperpigmentation, in particular, a skin whitening effect.

<44>

Previously, candidates such as natural products, extracts thereof, or chemical synthetic materials were treated only with melanocytes to evaluate the whitening efficacy. According to this conventional method, the candidate substance grafted in the cell culture should be treated directly to the cell, and the melanocytes cannot be treated with the candidate substance applied to the actual formulation, such as cream or lotion. Therefore, candidates can only be clinically tested using conventional methods. It can be determined whether the formulation containing the whitening substance has a clinically whitening effect. On the other hand, the skin whitening substance screening system according to the present invention can evaluate the presence or absence of the whitening effect by using the candidate substance in the culture medium as in the conventional method, as well as using a formulation such as cream or lotion containing the candidate substance. You can also evaluate the whitening effect. At this time, the skin whitening material screening system for evaluating the whitening effect on the formulation is made of artificial skin by laminating a plurality of layers including at least one of fibroblasts, melanocytes and keratinocytes and then exposing to air. Can be. As described above, the skin whitening screening system according to the present invention has a physiological and chemical characteristic similar to that of actual skin, and the evaluation of the whitening effect can be performed using a formulation that is actually used. It allows for efficient and simple sorting in less time and at less cost.

<46>

One aspect of the present invention is directed to one or more of WIF 1, WNT 1, WNT 5a and NFATC 2 of at least one of fibroblasts, keratinocytes and melanocytes in the skin lightening material screening system treated with the candidate substance. It provides a skin whitening material screening method comprising the step of checking the degree of expression or phosphorylation. As discussed above, WIF 1, WNT 1, WNT 5a and NFATC 2 are all related to the expression and diffusion of melanin pigment. Specifically, decreased expression of WIF 1, increased expression of TOT 1 or WNT 5a, or increased dephosphorylation of NFATC 2 resulted in an increase in the expression of tyrosinase, an enzyme involved in melanogenesis, and keratinocytes. This means an increase in melanin diffusion into the furnace. Therefore, by identifying how the candidate substance affects the expression level or phosphorylation degree of one or more of WIF 1, WNT 1, WNT 5a and NFATC 2, it is possible to evaluate whether the candidate substance has a skin whitening effect.

The method for screening a skin whitening substance according to an aspect of the present invention may further include determining a substance for increasing the expression level of WIF 1 as a skin whitening substance after confirming the expression level of WIF 1. Can be. In accordance with one aspect of the present invention, a method for screening a skin whitening material is provided, after determining the expression level of wr 1 or WNT 5a, determining a material that reduces the expression level of WNT 1 or WNT 5a as a skin whitening material. It may further include. According to another aspect of the present invention, a method for screening a skin whitening substance may further include determining a substance that increases the phosphorylation degree of NFATC 2 as a skin whitening substance after determining the phosphorylation degree of NFATC 2. Can be. Scoop Skin whitening substance screening system comprising at least one of fibroblasts, melanocytes and keratinocytes, which have reduced expression of WIF.l, increased expression of WNT 1 or WNT 5a, or dephosphorylation of FATC 2. Since it is reproduced in the actual skin condition where the skin degradation was performed, how the candidate substance changes the degree of expression of the WIF 1, WNT 1 or WNT 5a gene or the phosphorylation of the signal transducing factor NFATC 2 in the above-regulated system. By evaluating whether the candidate substance is a substance having a whitening effect, in particular, whether the substance can improve skin hyperpigmentation.

<49>

One aspect of the present invention provides a composition for skin whitening comprising a material screened according to the skin whitening material screening method as an active ingredient. As previously discussed, in one or more of the fibroblasts, keratinocytes and melanocytes

Substances that increase the expression of WIF 1, decrease the expression of WNT 1 or WNT 5a, or increase the degree of dephosphorylation of NFATC 2 may be considered to have a skin whitening effect, specifically, a substance that can improve skin hyperpigmentation. As such, a composition comprising the same as an active ingredient may exhibit a skin whitening effect, specifically, a skin hyperpigmentation improvement effect.

Another aspect of the present invention provides a skin whitening composition or an external skin composition comprising WIF 1 itself, which is a gene according to the present invention, thereby inhibiting the expression of tyrosinase of WIF 1. It may have an effect of improving skin whitening and hyperpigmentation by inhibiting melanin diffusion into keratinocytes.

According to another aspect of the present invention, a composition for skin whitening includes a cosmetic composition, a pharmaceutical composition, or a food composition.

<53>

Examples of the formulation of the composition according to one aspect of the present invention will be described below, but can also be used in various other formulations, which are intended to be described in detail only and not intended to limit the present invention.

<55>

<56> Formulation Example 1 Nutritional Cosmetics

According to the composition shown in Table 1 it can be prepared by the conventional method.

<58> [Table 1]

<59>

<60> [Formulation Example 2] Nutritional Lotion

According to the composition shown in Table 2 below it can be produced in a nutritious recipe.

<62> [Table 2]

<63>

<64> [Formulation Example 3] Nutrition Cream

Nutritional creams may be prepared by conventional methods according to the compositions shown in Table 3 below. <66> [Table 3]

<67>

<68> [Formulation Example 4] Pack

Packs can be prepared by conventional methods according to the compositions shown in Table 4 below.

<70> [Table 4]

<71>

[Formulation Example 5] Ointment

Ointments can be prepared by conventional methods according to the compositions shown in Table 5 below.

<74> [Table 5]

<75>

[Form for implementation of invention]

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to experimental examples, examples and comparative examples. However, the following Experimental Examples, Examples and Comparative Examples are provided only for the purpose of illustration in order to help the understanding of the present invention, but the scope and scope of the present invention is not limited thereto.

<77>

Experimental Example 1 Changes in WIF 1 Gene Expression in Skin Hyperpigmentation Sites

<79> Biopsy of 13 normal skin tissues (N) and skin tissues of hyperpigmentation site (L) of 13 subjects

RNA was extracted from the tissues by using biopsy, and gene expression changes were examined using real-time PCR. The results are shown in FIG.

As can be seen in Figure 1, the expression of WIF 1 in the skin tissue of the hyperpigmentation site is reduced compared to normal skin tissue. This suggests that WIF 1 is associated with skin hyperpigmentation.

<81>

Experimental Example 2 Changes in TOT 1 and TOT 5a Expression at the Skin Hyperpigmentation Site

<83> Biopsy of 13 normal skin tissues (N) and skin tissues (L) of hyperpigmentation sites was performed to extract RNA from tissues and to investigate the expression changes of WNT 1 and WNT 5a genes by real-time PCR. It was. The results are shown in FIG.

As can be seen in Figure 2, the TOT 1 and TOT 5a in the skin tissue of the hyperpigmentation site Expression is increased compared to normal skin tissue. This confirms that WNT 1 and WNT 5a are associated with skin hyperpigmentation.

<85>

Experimental Example 3 WIF 1 Gene Expression in Skin Cells

<87> Medium from Cascade Biologies

Into 254 (Medium 254) add human melanocyte growth supplement (HMGs) containing 10 ng / ml of phorbol 12-myri state 13-acetate (PMA), It's a badge _. Human skin melanocytes isolated from the epidermis of newborns were incubated in 37 ° C., 5% CO ₂ incubator. In addition, human keratinocyte growth supplement (HKGS human keratinocyte growth supplement) was added to medium EpiLife (# M-EPI-500-CA) obtained from Cascade Virologics, and isolated from the epidermis of newborn. Skin keratinocytes were incubated in 37 ^° C, 5% C0 ₂ incubator. Human dermal fibroblasts isolated from the epidermis of newborns were placed in DMEM (Dulbecco's Modified Eagles medium) containing 10% fetal bovine serum and cultured under 37 ^° C 5% C0 ₂ conditions. RNA was isolated from each of the skin cells cultured as described above, and the gene expression of WIF 1 was examined by real-time PCR, and the results are shown in FIG. 3.

As shown in FIG. 3, WIF 1 was expressed in fibroblasts (FB) and keratinocytes (KC), but not in melanocytes (MC). Through this, it can be seen that WIF 1 is expressed in fibroblasts and keratinocytes, but not melanocytes.

<89>

EXAMPLES 1-3 AND COMPARATIVE EXAMPLES 1 AND 2 PREPARATION OF THE SYSTEM

<9i> Significant composition of the dermis. 2.5 x in collagen Kcollagen I 1 mg / ml) solution

10 ⁵ / ml of fibroblasts were put in a 6-well plate, 2 ml per well, and then solidified in a 37 ^° C incubator for at least one hour to prepare a three-dimensional artificial dermis. A total of 2.5 x 10 ⁵ melanocytes and keratinocytes were mixed on a 1: 1 basis to prepare a three-dimensional system. At this time, the system using fibroblasts which suppressed the expression of WIF 1 by treating siRNA was set as Example 1, and the system using normal fibroblasts of human skin was set as Comparative Example 1.

And siRNA treatment in substantially the same manner as in Example 1 A three-dimensional system prepared by using keratinocytes and melanocytes in which strings were suppressed was used as Example 2, and a system prepared using normal keratinocytes and melanocytes was used as Comparative Example 2.

In addition, after treatment with WIF 1 made from a human recombinant gene at 50 ng / ml in the culture medium was mixed keratinocytes with melanocytes with increased expression of WIF1 was prepared in substantially the same manner as in Example 1 A system prepared by winding on one artificial dermis was used as Example 3.

<94>

Experimental Example 4 Evaluation of a System Including Fibroblasts Inhibiting WIF 1 Expression

Whether or not Example 1, a system according to the present invention, reflects well the actual skin condition was evaluated in comparison with Comparative Example 1 as follows.

<97> The system of Example 1, which contains fibroblasts that inhibited the expression of WIF 1, had a higher level of expression of tyrosinase, an enzyme that plays an important role in melanogenesis, as compared to the actual skin hyperpigmentation site. Significant increase was confirmed by Western blotting method through protein electrophoresis (see FIG. 4). In addition, staining keratinocytes with fluorescent antibodies that recognize keratin 14, which is expressed only in keratinocytes, and tyrosinase-related protein Ktyrosinase-related protein 1, TRP, expressed in melanocytes. 1) staining melanocytes with an antibody of different fluorescence that recognizes 1), and confirming the number of keratinocytes having both fluorescence using FACS (Fluorescence-activated cell sorting) Melanin diffusion into keratinocytes was increased compared to Comparative Example 1 as in the sub-pigmentation site (see FIG. 5). In addition, the phosphorylated form of the signal transduction factor NFATC 2 (nuclear factor of activated steel is, cytoplasmic, calcineurin-dependent 2) in the system of Example 1 by Western blotting method through protein electrophoresis (p-NFATC 2 ) Was decreased (see FIG. 6).

Based on the fibroblast melanocytes and keratinocytes laminated on the basis of the three-dimensional manufacturing system according to the present invention is similar to the actual skin condition, it can be seen that the skin hyperpigmentation site can be reproduced .

<100>

<ιοι> [Experiment 5] Evaluation of a system containing keratinocytes inhibited the expression of WIF 1 Whether or not Example 2, a system according to the present invention, reflects the actual skin condition was evaluated in comparison with Comparative Example 2 as follows.

<i03> Evaluation of the system of Example 2 including keratinocytes inhibiting the expression of WIF 1 was carried out in substantially the same manner as in Experiment 4, and thus played an important role in melanin production as in the actual skin hyperpigmentation site. It was confirmed that the expression of tyrosinase, an enzyme, was significantly increased compared to Comparative Example 2 (see FIG. 7).

In addition, evaluation in substantially the same manner as in Experimental Example 4, it was confirmed in Example 2 that melanin diffusion is increased like the skin hyperpigmentation site (see Fig. 8). In this case, it was confirmed that p-NFATC 2, a phosphorylated form of NFATC 2, was reduced.

It was confirmed that NFATC 2 was dephosphorylated (see FIG. 9).

Through this, the system according to the present invention manufactured in three dimensions using melanocytes and keratinocytes is similar to the actual skin condition, and it can be confirmed that the skin hyperpigmentation site can be reproduced.

<106>

Experimental Example 6 Evaluation of a System Containing Melanosite Increased Expression of Cells by Directly Treating WIF 1 Made with Human Recombinant Gene in Culture Medium

<108> WIF 1 was processed by Western blotting using protein electrophoresis for the system of Example 3 comprising melanocytes treated with WIF 1 made using a human recombinant gene to be 50 ng / ml in the cell culture medium. It was confirmed that the expression of increased (see Figure 10). In this case, it was confirmed that the expression of tyrosinase was reduced in the system of Example 3 in substantially the same manner as in Experimental Example 4 (see FIG. 11). In addition, as compared with Comparative Example 1, it was confirmed that the melanin diffusion in Example 3 is reduced (see Fig. 12), it was also confirmed that the phosphorylation of NFATC 2 is increased (see Fig. 13).

Through this process, WIF 1 and NFATC 2 are closely related to melanin pigment production, and thus the candidate material is treated in the system according to the present invention, and then the expression level of WIF 1 and its lower signaling factor NFATC 2 are reduced. By assessing the degree of phosphorylation, it can be seen that the skin whitening material can be screened.

<110>

As described above, the skin whitening material screening system according to the present invention can reproduce the actual skin, thereby effectively screening the material exhibiting clinically excellent skin whitening effect even in vitro. Furthermore, this is the actual skin tone of human Because it reflects the job well, it can be used to study the mechanism or improvement of pigment deficiency such as vitiligo or white hair.

Claims

[Range of request]

[Claim 1]

A skin lightening material screening system comprising one or more layers comprising one or more cells of fibroblasts, keratinocytes and melanocytes.

[Claim 2]

The method of claim 1,

A fibroblast layer comprising fibroblasts; And

A skin whitening material screening system comprising a layer comprising at least one of keratinocytes and melanocytes.

[Claim 3]

The method of claim 2,

Fibroblasts are skin whitening substance screening systems, which are layers containing fibroblasts in collagen.

[Claim 4]

The method of claim 3,

Collagen skin whitening material screening system comprises a skin whitening material screening system in a 0.001 increase ¾> to 30 weight ¾ based on the total weight of the skin whitening material screening system.

[Claim 5]

The method of claim 1,

At least one of fibroblasts, keratinocytes and melanocytes is a skin whitening substance screening system in which the expression of WIF KWnt inhibitory factor 1) is inhibited.

[Claim 6]

The method of claim 1,

At least one of fibroblasts, keratinocytes and melanocytes are cells with increased expression of WNT 1 or WNT5a.

[Claim 7]

The method of claim 1,

. Skin whitening material screening system wherein at least one of fibroblasts, keratinocytes and melanocytes is a cell dephosphorylated with NFATC 2 (nuclear factor of activated T cells, cytoplasmic 2).

[Claim 8]

According to claim 1, ^■

Skin whitening material screening system wherein at least one of the fibroblasts, keratinocytes and melanocytes is a cell derived from at least one selected from human, mouse, guinea pig, chicken and swine diseases.

[Claim 9]

According to claim 1,

Skin whitening material screening system comprising a whitening material to improve skin hyperpigmentation.

[Claim 10]

In the skin whitening substance screening system according to any one of claims 1 to 9, wherein the candidate substance has been treated, WIF 1, WNT 1, WNT 5a and NFATC 2 of at least one of fibroblasts, keratinocytes and melanocytes. A method for screening a skin whitening material comprising determining the degree of expression or phosphorylation of at least one.

[Claim 111]

The method of claim 10,

After the step of determining the degree of expression or phosphorylation, skin whitening material screening method further comprising the step of determining the substance to increase the expression level of WIF 1 as a skin whitening material.

[Claim 12]

The method of claim 10, After the step of determining the degree of expression or phosphorylation, skin whitening material screening method further comprising the step of determining the substance to reduce the expression level of WNT 1 or WNT 5a as a skin whitening material.

[Claim 13]

The method of claim 10,

After the step of determining the degree of expression or phosphorylation, whitening substance screening method further comprising the step of determining the substance to increase the phosphorylation degree of NFATC 2 as a skin whitening substance.

[Claim 14]

A composition for skin whitening comprising a substance screened according to the screening method of claim 10 as an active ingredient.

[Claim 15]

Skin whitening composition comprising WIF 1 as an active ingredient. [Claim 16]

An external preparation composition for skin containing WIF 1 as an active ingredient. [Claim 17]

The composition according to claim 15 or 16, wherein the composition is a cosmetic composition.