JP6069334B2 - Skin whitening substance screening structure - Google Patents

Description

  The present invention relates to a skin whitening substance screening system and a skin whitening substance screening method.

  So far, in developing new skin whitening substances, after treating candidate substances directly to melanocytes, which are melanocytes, whether to suppress the production of melanin or irradiating melanocytes with ultraviolet rays The method has been used to check whether the candidate substance blocks the melanin production signal generated at that time. However, in fact, melanocytes are not present alone in the skin, but are directly or indirectly affected by physical or chemical influences from other surrounding cells and the microenvironment. It is not possible to reproduce such actual skin tissue condition by itself.

  In addition, skin hyperpigmentation such as spots, spots, and moles is different from the increase in melanin production due to ultraviolet rays, although the cause and mechanism of the skin hyperpigmentation tend to be worsened by ultraviolet rays, but is not stimulated by ultraviolet rays. In some cases, the production and dispersion of melanin continues to increase. Therefore, skin whitening substances developed based on existing ultraviolet irradiation methods often do not show the effect of suppressing human skin hyperpigmentation in actual clinical practice. In addition, to study skin hyperpigmentation, biopsying and using the skin of a subject's hyperpigmentation site is very expensive and labor intensive and can produce human skin tissue In addition to the UV irradiation model, it is difficult to obtain an animal model of cutaneous hyperpigmentation.

  Therefore, there is a need for a system and a screening method that can easily screen skin whitening substances while being very similar to the structure of human skin tissue, particularly human skin tissue exhibiting skin hyperpigmentation. doing.

Korean Registered Patent No. 10-0840144

  One aspect of the present invention is to provide a skin whitening substance screening system capable of screening clinically useful skin whitening substances while being effective, and a skin whitening substance screening method using the same. Is.

  Another aspect of the present invention is to provide a composition for skin whitening which contains a substance screened by the skin whitening substance screening method as an active ingredient and exhibits an excellent whitening effect.

  Another aspect of the present invention is to provide a skin whitening composition or a skin external preparation composition containing a skin melanin production-related gene itself as an active ingredient.

  One aspect of the present invention provides a skin whitening substance screening system including one or more layers including one or more of fibroblasts, keratinocytes, and melanocytes.

  According to another aspect of the present invention, in the skin whitening substance screening system in which a candidate substance is treated, one or more of WIF1, WNT1, WNT5a, and NFATC2 of one or more cells among fibroblasts, keratinocytes, and melanocytes. A method for screening a skin lightening substance, comprising the step of confirming the degree of expression or the degree of phosphorylation.

  Another aspect of the present invention is a skin whitening composition comprising as an active ingredient a substance screened by the skin whitening substance screening method, or a skin whitening composition and an external preparation for skin containing WIF1 itself as an active ingredient. A composition is provided.

  Since the skin whitening substance screening system according to the present invention is designed close to the actual skin tissue, it is possible to easily screen for substances having a clinical whitening effect in vitro with less time and cost. In particular, the skin whitening substance screening system according to the present invention is different from an existing method in which a whitening substance is screened by irradiating UV light after treating a candidate substance on melanocytes, and the actual skin showing hyperpigmentation. Since it has conditions similar to the degree of tissue gene expression and phosphorylation of signal transduction factors, skin whitening substances, especially substances effective for improving hyperpigmentation, can be easily screened when using them. can do. This is useful for developing new skin lightening substances, specifically hyperpigmentation-improving substances in that it is difficult to obtain human and animal models for hyperpigmentation research. System or kit.

It is the graph which compared the difference of the expression level of a WIF1 gene of a test subject's normal skin (N) and a skin hyperpigmentation site | part (L). It is the graph which compared the difference of the expression level of WNT1 and WNT5a of a test subject's normal skin (N) and a skin hyperpigmentation site | part (L). It is the graph which compared the difference in the expression level of WIF1 gene in a melanocyte (MC), a keratinocyte (KC), and a fibroblast (FB). It is a result which shows that the expression of tyrosinase increases in the whitening screening system containing the fibroblast which suppressed the expression of WIF1. It is a result which shows that the movement of the melanosome increased in the whitening screening system containing the fibroblast which suppressed the expression of WIF1. It is a result which shows that NFATC2 is dephosphorylated in the whitening screening system containing the fibroblast which suppressed the expression of WIF1. It is an experimental result which shows that the expression of tyrosinase increases in the whitening screening system containing the keratinocyte which suppressed the expression of WIF1. It is a result which shows that the movement of melanosome increased in the whitening screening system containing the keratinocyte which suppressed the expression of WIF1. It is a result which shows that NFATC2 is dephosphorylated in the whitening screening system containing the keratinocyte which suppressed the expression of WIF1. It is a result which shows that WIF1 expression of a melanocyte increases when the human WIF1 produced by the gene recombination technique is processed into a melanocyte. FIG. 11 is a result showing that the expression of tyrosinase decreases in melanocytes in which the expression of WIF1 is induced as shown in FIG. It is a result which shows that the movement of melanosome decreased when human WIF1 produced by the gene recombination technique was processed in a whitening screening system containing melanocytes. It is a result which shows that NFATC2 is phosphorylated in the melanocyte which induced the expression of WIF1 like FIG.

  In the present specification, “skin” means a tissue covering the body surface of an animal, and is a broadest concept including not only the tissue covering the body surface such as the face or body but also the scalp and hair. is there.

  Skin melanocytes are cells that make melanin that determines skin color and are located on the basement membrane, which acts as the boundary between the dermis and epidermis of the skin. Developmentally, melanocytes start from neural-crest cells, migrate to the skin via melanocyte stem cells and progenitor cells, melanoblasts, and finally It is known to differentiate into melanocytes, which are pigment cells capable of producing melanin.

  The differentiated melanocytes do not exist alone in the skin, but are affected by the binding of keratinocytes, which are keratinocytes located above them, and fibroblasts in the dermis located below them. Will be generated. Melanin made from melanocytes moves to the surrounding keratinocytes via cell dendrites and is widely dispersed on the skin surface, thus protecting the skin from harmful factors such as ultraviolet rays and developing the skin color. . Normal skin produces more melanin than normal when stimulated by UV rays, increases melanin dispersion, and then disappears once again when the stimulus disappears. return.

  As mentioned earlier, it does not reflect the actual skin condition that melanocytes produce melanin under the combined influence of physical and chemical factors of other cells and surrounding microenvironment in the skin. Whitening substances developed by existing methods, especially skin whitening substances developed by ultraviolet irradiation methods, often have a clinically low effect in improving hyperpigmentation of the skin. Skin hyperpigmentation is a darkening of the skin due to various causes and specific mechanisms. To develop a whitening substance that can effectively improve this, it is similar to that of actual skin. It is necessary to develop a whitening substance using this experimental model.

  Hereinafter, the present invention will be described in detail.

  One aspect of the present invention provides a skin whitening substance screening system including one or more layers including one or more of fibroblasts, keratinocytes, and melanocytes. Another aspect of the present invention provides a skin whitening substance screening system including a fibroblast cell layer including fibroblasts; a keratinocyte layer including keratinocytes and a melanocyte layer including melanocytes. At this time, the fibroblast layer may be laminated at the bottom, the melanocyte layer may be laminated thereon, and the keratinocyte layer may be laminated thereon, similar to actual skin conditions.

  A skin whitening substance screening system according to another aspect of the present invention includes a fibroblast layer containing fibroblasts; and a layer containing one or more of keratinocytes and melanocytes, wherein the keratinocytes and melanocytes are the same. May be included in the layer. At this time, a fibroblast layer may be laminated below, and a layer containing melanocytes and keratinocytes may be laminated thereon. The skin whitening substance screening system is a three-dimensional structure system in which skin cell layers are stacked, and can accurately reflect the actual skin condition by including not only melanocytes but also surrounding cells. The skin lightening substance screening system according to another aspect of the present invention may include a layer containing melanocytes and keratinocytes, which are known to play an important role in the production of melanin, except for the fibroblast layer.

  The fibroblast layer of the skin whitening substance screening system according to one aspect of the present invention reproduces the dermis layer and includes a layer in which fibroblasts are contained in collagen. Specifically, the fibroblast layer may be a layer produced by putting fibroblasts in a collagen solution and solidifying them.

  In another aspect of the present invention, the collagen is 0.001% to 30% by mass, specifically 0.01% to 20% by mass, more specifically based on the total weight of the skin whitening substance screening system. In particular, 0.1% by mass to 10% by mass may be contained. When included in the above range, it is suitable for showing the intended effect of the present invention, and it may be appropriate to use in the above range also in terms of cost effectiveness.

  In the skin whitening substance screening system according to one aspect of the present invention, one or more cells among fibroblasts, keratinocytes and melanocytes are one or more selected from birds such as humans, mice, guinea pigs and chickens, and pigs. Cells derived from.

  The skin whitening substance screening system according to one aspect of the present invention is not only a substance having a skin whitening effect, specifically, a substance having an effect of improving skin blackening by ultraviolet rays, but particularly, skin hyperpigmentation such as spots, spots, and moles. Substances having an effect of improving symptoms may be effectively screened.

  The present inventors have expressed the expression of WIF1 (Wnt inhibitory factor 1, ACCESSION NP_009122, VERSION NP_009122.2 GI: 111125011) gene, which is an extracellular signaling molecule associated with embryo growth regulation, in skin showing hyperpigmentation. Specifically, the expression of WIF1 gene affects melanin production of melanocytes by decreasing in keratinocytes and fibroblasts, not melanocytes, which are melanocytes, among skin cells. Revealed. Moreover, in the skin where the expression of WIF1 gene decreased, the expression of tyrosinase involved in the production of melanin is increased, and at the same time, the diffusion of melanin to keratinocytes is increased. It was confirmed that there was a close relationship. At this time, the expression of WIF1 gene is involved in the expression of GSK-3β (Glycogen synthase kinase-3β) and β-catenin phosphorylation, MITF (microphthalmia-associated transcription factor), and the overexpression of the pigment. It was also confirmed that it affects the deposition.

  Accordingly, one aspect of the present invention is a skin whitening substance screening system in which WIF1 expression of one or more cells among fibroblasts, keratinocytes and melanocytes is suppressed, specifically, WIF1 expression of keratinocytes or fibroblasts is suppressed. An improved skin lightening substance screening system is provided. In another aspect of the present invention, the skin whitening substance screening system includes cells in which the expression of WIF1 is suppressed using siRNA. The screening system reflects a hyperpigmented skin condition in which the expression of WIF1 in keratinocytes or fibroblasts is reduced, and may reproduce a skin condition exhibiting hyperpigmentation almost similar to the actual condition. Therefore, when using the screening system, a substance having a skin whitening effect, particularly a substance effective in improving skin hyperpigmentation, may be effectively selected.

  Further, the present inventors have also shown that proto-oncogene precursor WNT1 (Wingless-type MMTV integration site family, member 1, ACCESSION NP_005421, VERSION NP_005421.1G in skin showing hyperpigmentation. ) And protein WNT5a (Wingless-type MMTV integration site family, member 5a, ACCESSION NP_0012430304, VERSION NP_0012433034.1 GI: 371502087) cells, cytoplasmic, calcineurin-dependent 2) are dephosphorylated, and therefore, one aspect of the present invention is that one or more of fibroblasts, keratinocytes, and melanocytes are expressed by WNT1 and WNT5a. A skin whitening substance screening system in which increased cells or NFATC2 is dephosphorylated is provided. Since the screening system can actually reproduce the skin condition showing the hyperpigmentation layer, it effectively selects substances that have a skin whitening effect, in particular, substances that are effective in improving skin hyperpigmentation. It's okay.

  Conventionally, a candidate substance such as a natural product, an extract thereof, or a chemically synthesized substance is treated only on melanocytes to evaluate the whitening effect. In the case of such existing methods, the candidate substance diluted in the cell culture medium must be processed directly into the cells, and in the state where the candidate substance is applied to the actually used dosage form such as cream or lotion. Can not be processed into melanocytes. Therefore, with existing methods, it is not until clinical trials are conducted that it is possible to confirm whether a dosage form containing a candidate whitening substance actually has a whitening effect clinically.

  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 diluting and using the candidate substance in the culture solution as in the existing method. The presence or absence of the whitening effect can also be evaluated using a dosage form such as a cream or lotion containing a candidate substance. At this time, a skin whitening substance screening system for evaluating the presence or absence of a whitening effect on a dosage form is formed by laminating a plurality of layers including one or more of fibroblasts, melanocytes and keratinocytes, and then exposing them to air. Or manufactured as artificial skin.

  As described above, the skin whitening screening system according to the present invention has physiological and chemical characteristics similar to those of actual skin, and the whitening effect can be evaluated even when a dosage form actually used is used. Therefore, it is possible to effectively and effectively sort out substances that have a clinical whitening effect in a shorter time and at a lower cost.

  One aspect of the present invention is the above skin whitening substance screening system in which a candidate substance is treated, wherein one or more of fibroblasts, keratinocytes and melanocytes are expressed in one or more of WIF1, WNT1, WNT5a and NFATC2. A method for screening a skin whitening substance, comprising the step of confirming the degree of phosphorylation or the degree of phosphorylation. As mentioned earlier, WIF1, WNT1, WNT5a and NFATC2 are all associated with the degree of melanin pigment expression and diffusion in the skin. Specifically, decreasing the expression of WIF1, increasing the expression of WNT1 or WNT5a, or increasing the degree of dephosphorylation of NFATC2 increases the expression of tyrosinase, an enzyme involved in melanin production. This means that melanin diffusion to keratinocytes is increased. Therefore, by confirming how the candidate substance affects the level of expression or phosphorylation of one or more of WIF1, WNT1, WNT5a and NFATC2, whether the candidate substance has a skin whitening effect You can evaluate whether.

  The skin whitening substance screening method according to one aspect of the present invention may further include a step of determining a substance that increases the expression level of WIF1 as a skin whitening substance after the step of confirming the expression level of WIF1. The skin whitening substance screening method according to another aspect of the present invention includes a step of determining a substance that decreases the expression level of WNT1 or WNT5a as a skin whitening substance after the step of confirming the expression level of WNT1 or WNT5a. Further may be included.

  The skin whitening substance screening method according to another aspect of the present invention further includes a step of determining a substance that increases the degree of phosphorylation of NFATC2 as a skin whitening substance after the step of confirming the degree of phosphorylation of NFATC2. It's okay. In particular, screening for skin whitening substances comprising one or more of fibroblasts, melanocytes and keratinocytes in which WIF1 expression is decreased, WNT1 or WNT5a expression is increased, or NFATC2 is dephosphorylated. Since the system reproduces the actual skin condition where skin darkening has been achieved, the degree of expression of the WIF1, WNT1 or WNT5a gene of the system in which the candidate substance is regulated as described above or the signaling factor NFATC2 To determine whether the candidate substance is a substance that has a whitening effect, in particular, whether it can improve skin hyperpigmentation be able to.

  One aspect of the present invention provides a composition for skin whitening comprising, as an active ingredient, a substance screened by the skin whitening substance screening method. As described above, in one or more of fibroblasts, keratinocytes, and melanocytes, the expression of WIF1 is increased, the expression of WNT1 or WNT5a is decreased, or the degree of dephosphorylation of NFATC2 is increased. Since the substance to be increased can be judged to be a skin whitening effect, specifically, a substance having an effect of improving skin hyperpigmentation, a composition containing this as an active ingredient has a skin whitening effect, specifically In addition, skin hyperpigmentation can be improved.

  Another aspect of the present invention is to provide a whitening composition or an external skin composition containing WIF1 itself, which is a gene according to the present invention as described above, thereby suppressing the expression of tyrosinase of WIF1 and keratinocytes. It can show skin whitening and skin hyperpigmentation improving effects by suppressing melanin diffusion into the skin.

  The whitening composition according to another aspect of the present invention includes a cosmetic composition, a pharmaceutical composition, or a food composition.

  Examples of dosage forms of the composition according to one aspect of the present invention will be described below, but the present invention can also be applied to various other dosage forms, and these are not intended to limit the present invention, but merely specifically. It is for explanation.

[Formulation Example 1] Nutritional lotion According to the composition shown in Table 1 below, a nutritional lotion may be produced by an ordinary method.

[Formulation Example 2] Nutrition Lotion According to the composition described in Table 2 below, a nutrition lotion may be produced by a normal method.

[Formulation Example 3] Nutritional Cream According to the composition described in Table 3 below, a nutritional cream may be produced by a normal method.

[Formulation Example 4] Pack According to the composition described in Table 4 below, a pack may be produced by a usual method.

[Formulation Example 5] Ointment According to the composition described in Table 5 below, an ointment may be produced by an ordinary method.

  Hereinafter, the configuration and effects of the present invention will be described more specifically 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 to aid the understanding of the present invention, and the scope and scope of the present invention are limited thereby. It is not something.

[Experimental example 1] Expression change of WIF1 gene in skin hyperpigmentation site Biopsy of 13 subjects' normal skin tissue (N) and hyperpigmentation site skin tissue (L) to extract RNA in tissue Using this, changes in gene expression were investigated by a real-time PCR method. The results are shown in FIG.

  As shown in FIG. 1, the expression of WIF1 is decreased in the skin tissue at the hyperpigmentation site as compared with the normal skin tissue. Through this, it can be confirmed that WIF1 is associated with skin hyperpigmentation.

[Experimental example 2] Expression change of WNT1 and WNT5a in skin hyperpigmentation site Biopsy of normal skin tissue (N) and skin tissue (L) of hyperpigmentation site of 13 subjects, RNA in the tissue was extracted, Using this, changes in the expression of WNT1 and WNT5a genes were investigated by a real-time PCR method. The results are shown in FIG.

  As shown in FIG. 2, the expression of WNT1 and WNT5a is increased in the skin tissue at the hyperpigmented site as compared to the normal skin tissue. Through this, it can be confirmed that WNT1 and WNT5a are associated with skin hyperpigmentation.

[Experimental Example 3] WIF1 gene expression in skin cells Medium 254 obtained from Cascade Biologics was added to phorbol 12-myristate 13-acetate (PMA, 10 ng / mg of phorbol 12-myristate 13-acetate). Human skin melanocytes separated from neonatal epidermis were cultured in a 37 ° C., 5% CO ₂ incubator using human melanocyte growth supplements (HMGs, human supplement growth supplement) containing ml. In addition, medium EpiLife (# M-EPI-500-CA) obtained from Cascade Biologics, Inc. is filled with human keratinocyte growth supplement (HKGS, human keratinocyte growth supplement) and used as a medium to separate from the epidermis of the newborn The human skin keratinocytes were cultured in a 37 ° C., 5% CO ₂ incubator. Human skin fibroblasts separated from neonatal epidermis were placed in DMEM (Dulbecco's Modified Eagles medium) containing 10% fetal bovine serum, and cultured under conditions of 37 ° C. and 5% CO ₂ . RNA was isolated from each skin cell cultured as described above, and the gene expression of WIF1 was investigated by real-time PCR. The results are shown in FIG.

  As shown in FIG. 3, WIF1 was expressed in fibroblasts (FB) and keratinocytes (KC), but not in melanocytes (MC). Through this, it can be known that WIF1 is expressed not in melanocytes but in fibroblasts and keratinocytes in skin cells.

[Examples 1 to 3 and Comparative Examples 1 to 2] System Production 2.5 x 10 ⁵ / ml fibroblasts were placed in a collagen 1 (collagen I, 1 mg / ml) solution, which is an important component of the dermis. Then, 2 ml per well was placed in a 6-well plate, and then solidified in a 37 ° C. incubator for 1 hour or more to prepare a three-dimensional artificial dermis. On top of that, a total of 2.5 × 10 ⁵ melanocyte and keratinocyte layers mixed 1: 1 were placed to produce a three-dimensional system. At this time, a system using fibroblasts treated with siRNA to suppress the expression of WIF1 was set as Example 1, and a system using normal fibroblasts of human skin was set as Comparative Example 1.

  A three-dimensional system prepared using keratinocytes and melanocytes, in which siRNA was treated in substantially the same manner as in Example 1 to suppress the expression of WIF1, was used as Example 2, and normal keratinocytes and melanocytes were used. The system produced in this way was referred to as Comparative Example 2.

  Then, after treating WIF1 produced using a human recombinant gene in a culture medium at 50 ng / ml and mixing the melanocytes with increased expression of WIF1, keratinocytes were mixed in substantially the same manner as in Example 1. A system manufactured on the manufactured artificial dermis was referred to as Example 3.

[Experimental Example 4] Evaluation for a system including fibroblasts in which the expression of WIF1 is suppressed As to whether Example 1 which is a system according to the present invention correctly reflects an actual skin condition, Comparative Example 1 is as follows. It was evaluated while contrasting.

  In the system of Example 1 including fibroblasts in which the expression of WIF1 is suppressed, the expression of tyrosinase, which is an enzyme that plays an important role in the production of melanin, is similar to that of the actual skin hyperpigmentation site. It was confirmed by the Western blotting method through protein electrophoresis that the increase was significantly higher than that of (see FIG. 4).

  Further, keratinocytes are stained with a fluorescently labeled antibody that recognizes keratin 14 expressed only in keratinocytes, and different colors for recognizing tyrosinase-related protein 1, TRP 1 expressed in melanocytes. As a result of confirming the number of keratinocytes having both two fluorescences using FACS (Fluorescence-activated cell sorting), Example 1 Like the hyperpigmentation site, melanin diffusion to keratinocytes was increased compared to Comparative Example 1 (see FIG. 5). At this time, the phosphorylated form of the signal transduction factor NFATC2 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2) in the system of Example 1 through the Western blotting method through protein electrophoresis (p. -It was possible to observe a decrease in NFATC2) (see Fig. 6).

  Based on these facts, the system according to the present invention, which is three-dimensionally manufactured by laminating fibroblasts, melanocytes and keratinocytes, is similar to the actual skin condition, and in particular, can reproduce the skin hyperpigmentation site. Can be confirmed.

[Experimental Example 5] Evaluation for a system containing keratinocytes in which the expression of WIF1 is suppressed Whether or not Example 2 which is a system according to the present invention correctly reflects an actual skin state is compared with Comparative Example 2 as follows. While evaluating.

  As a result of evaluating the system of Example 2 containing keratinocytes in which the expression of WIF1 was suppressed by substantially the same method as in Experimental Example 4, as with the actual skin hyperpigmentation site, it played an important role in the production of melanin. It was confirmed that the expression of tyrosinase, which is an enzyme that increases, was remarkably increased compared to Comparative Example 2 (see FIG. 7).

  Moreover, when it evaluated by the method substantially the same as Experimental example 4, also in Example 2, it confirmed that melanin diffusion was increasing like a skin hyperpigmentation site | part (refer FIG. 8). At this time, it was confirmed that p-NFATTC2, which is a phosphorylated form of NFATC2, was decreased, and that NFATC2 was dephosphorylated (see FIG. 9).

  Through these things, it can be confirmed that the system according to the present invention manufactured three-dimensionally using melanocytes and keratinocytes is similar to the actual skin state, and in particular, can reproduce the skin hyperpigmentation site.

[Experimental Example 6] Evaluation for a system containing melanocytes in which WIF1 produced using a human recombinant gene was directly treated in a culture medium to increase WIF1 expression in the cell itself. Regarding the system of Example 3 containing melanocytes treated with WIF1 in a cell culture medium to 50 ng / ml, it was confirmed that the expression of WIF1 was increased by Western blotting through protein electrophoresis (see FIG. 10). At this time, it was confirmed that the expression of tyrosinase decreased in the system of Example 3 by substantially the same method as in Experimental Example 4 (see FIG. 11). As a result of comparison with Comparative Example 1, it was confirmed that the diffusion of melanin was reduced in Example 3 (see FIG. 12), and it was also confirmed that phosphorylation of NFATC2 was increased (see FIG. 13).

  Through these facts, WIF1 and NFATC2 are closely related to the production of melanin pigment, so that the candidate substance of the system according to the present invention is processed, and then the degree of increased expression of WIF1 and its lower signaling factor It can be known that skin whitening substances can be screened through evaluation of the degree of phosphorylation of NFATC2.

  As described above, since the skin whitening substance screening system according to the present invention can reproduce the actual skin, it is possible to effectively screen substances exhibiting a clinically excellent whitening effect even in vitro. Furthermore, since it correctly reflects the actual human skin tissue, it can also be used to study the mechanisms of pigment deficiencies such as vitiligo or white hair or methods of amelioration.

Claims (9)

A fibroblast layer comprising fibroblasts ;
A keratinocyte (keratinocyte) and melanocytes (melanocyte) skin and including layers laminated whitening substance screening structure,
One or more of the fibroblasts, keratinocytes and melanocytes are compared to normal skin tissue,
Expression of WIF1 (Wnt inhibitory factor 1) is suppressed,
The expression of WNT1 or WNT5a is increased or NFATC2 (nuclear factor of activated T cells, cytoplasmic 2) is dephosphorylated,
Skin whitening substance screening structure . The skin whitening substance screening structure according to claim 1 , wherein the fibroblast layer is a layer in which fibroblasts are contained in collagen. The skin whitening substance screening structure according to claim 2 , wherein the collagen is contained in an amount of 0.001% by mass to 30% by mass based on the entire weight of the skin whitening substance screening structure . The skin whitening substance according to claim 1, wherein one or more cells among fibroblasts, keratinocytes and melanocytes are cells derived from one or more selected from human, mouse, guinea pig, chicken and pig. Screening structure . The skin whitening substance screening structure according to claim 1, wherein the skin whitening substance includes a skin whitening substance that improves skin hyperpigmentation. In skin-whitening agent screening structure according to any one of claim 1 to 5 which process the candidate substance, fibroblasts, of one or more cells of the keratinocytes and melanocytes WIF1, WNT1 and Wnt5 a least one of A method for screening a skin whitening substance, comprising a step of confirming the expression level of NFAT2 or the degree of dephosphorylation of NFATC2 . The skin whitening according to claim 6 , further comprising the step of determining a substance that increases the expression level of WIF1 as compared with normal skin tissue as a skin whitening substance after the step of confirming the degree of expression or the degree of phosphorylation. Substance screening method. The method of claim 6 , further comprising the step of determining a substance that decreases the expression level of WNT1 or WNT5a as a skin whitening substance as compared with normal skin tissue after the step of confirming the degree of expression or the degree of phosphorylation. Skin whitening substance screening method. The skin according to claim 6 , further comprising the step of determining a substance that increases the degree of phosphorylation of NFATC2 as compared with normal skin tissue as a skin whitening substance after the step of confirming the degree of expression or the degree of phosphorylation. Whitening substance screening method.