Method for screening inhibitor and / or improver of skin pigmentation symptom

Abstract

To provide a screening method for an inhibitor and / or an improver for skin pigmentation symptoms.SOLUTION: A screening method for an inhibitor and / or an improver for skin pigmentation symptoms uses a degree of contact between a nerve fiber and a melanocyte (Mc) as an index.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for screening an inhibitor and / or improver of skin pigmentation symptoms.

Background Art

[0002] Skin pigmentation symptoms such as freckles and chloasma greatly affect the appearance of the face, so there is a high interest in their prevention and improvement. Conventionally, whitening agents with various mechanisms of action have been developed, and consumers can select cosmetics according to their preferences and desired effects.

[0003] As a method for screening agents having skin pigmentation symptoms such as freckles and chloasma, Patent Document 1 discloses a method for screening a freckle-improving component, which is characterized by adding melanosomes to fibroblasts.

[0004] Further, Patent Document 2 discloses a method for screening an active ingredient that exhibits a freckle-improving effect and / or a whitening effect, which is characterized by measuring tyrosinase activity and cell activity.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] In view of the above prior art, an object of the present invention is to provide a novel method for screening an inhibitor and / or improver of skin pigmentation symptoms. In particular, an object of the present invention is to provide a novel method for screening a freckle inhibitor and / or a freckle improver.

Means for Solving the Problem

[0007] The screening method of the present invention for solving the above problems is a screening method for an inhibitor and / or ameliorant of skin pigmentation symptoms, using the degree of contact between nerve fibers and melanocytes (Mc) as an index.

[0008] In a preferred embodiment of the screening method of the present invention, it is characterized in that a candidate substance with a reduced degree of contact between nerve fibers and melanocytes (Mc) and / or a candidate substance for which suppression of melanocyte (Mc) contact of nerve fibers has been confirmed is determined to be a more excellent inhibitor and / or ameliorant of skin pigmentation symptoms.

[0009] In a preferred embodiment of the screening method of the present invention, it is characterized in that a candidate substance with a reduced expression level of PSD95 in the epidermis and / or a candidate substance for which an increase in the expression level of PSD95 in the epidermis has been suppressed is determined to be a more excellent inhibitor and / or ameliorant of skin pigmentation symptoms.

[0010] Further, in a preferred embodiment of the screening method of the present invention, the pigmentation symptom is a freckle.

[0011] Further, in a preferred embodiment of the screening method of the present invention, an inhibitor and / or ameliorant of skin pigmentation symptoms having an action of reducing the degree of contact between nerve fibers and melanocytes (Mc) and / or an action of suppressing melanocyte (Mc) contact of nerve fibers is used as a screening target.

[0012] Further, a preferred embodiment of the screening method of the present invention uses a device including a culture tank having two cell placement parts and a connecting part connecting the cell placement parts, a preparation step of placing nerve cells in one cell placement part, placing melanocytes (Mc) in the other cell placement part, and applying a candidate substance to nerve fibers and / or melanocytes (Mc), A measuring step of co-culturing nerve cells and melanocytes (Mc) and measuring the degree of contact between nerve fibers and melanocytes (Mc); Based on the results of the measuring step, a determination step of determining that a candidate substance with a decreased degree of contact between nerve fibers and melanocytes (Mc) and / or a candidate substance for which suppression of melanocyte (Mc) contact of nerve fibers has been confirmed is a better inhibitor and / or improver of skin pigmentation symptoms; characterized by comprising the above.

[0013] In a preferred embodiment of the screening method of the present invention, the measuring step includes measuring the contact ratio of melanocytes (Mc) and nerve fibers per predetermined area and / or measuring melanocytes (Mc) with a changed hue per predetermined area.

[0014] Also, a preferred embodiment of the screening method of the present invention is A preparation step of applying a candidate substance to nerve fibers and / or melanocytes (Mc); A measuring step of co-culturing nerve cells and melanocytes (Mc) and measuring the degree of contact between nerve fibers and melanocytes (Mc); Based on the results of the measuring step, a determination step of determining that a candidate substance with a decreased degree of contact between nerve fibers and melanocytes (Mc) and / or a candidate substance for which suppression of melanocyte (Mc) contact of nerve fibers has been confirmed is a better inhibitor and / or improver of skin pigmentation symptoms; characterized by comprising the above.

[0015] Also, a preferred embodiment of the screening method of the present invention is A preparation step of applying two or more candidate substances to two or more test systems including nerve fibers and melanocytes (Mc) respectively; A measuring step of co-culturing nerve cells and melanocytes (Mc) and measuring the degree of contact between nerve fibers and melanocytes (Mc); Based on the measurement results of the measurement step, a candidate substance with a reduced degree of contact between nerve fibers and melanocytes (Mc) and / or a candidate substance for which suppression of the contact between nerve fibers and melanocytes (Mc) has been confirmed are determined to be better inhibitors and / or improvers of skin pigmentation symptoms in a determination step. It is characterized by having

[0016] In a preferred embodiment of the screening method of the present invention, The measurement step includes measuring the co-localization of nerve cells and melanocytes (Mc). The determination step is characterized by determining the degree of contact between nerve fibers and melanocytes (Mc) based on the co-localization of nerve fibers and melanocytes (Mc).

[0017] In a preferred embodiment of the screening method of the present invention, The measurement step includes staining each of nerve fibers and melanocytes (Mc). The determination step is characterized by determining the degree of contact between nerve fibers and melanocytes (Mc) based on the coincidence rate of the staining positions of nerve fibers and melanocytes (Mc).

[0018] In a preferred embodiment of the screening method of the present invention, The measurement step includes measuring the expression level of PSD95. The determination step is characterized by determining that a candidate substance with a reduced expression level of PSD95 in the epidermis and / or a candidate substance for which the increase in the expression level of PSD95 in the epidermis is suppressed is a better inhibitor and / or improver of skin pigmentation symptoms.

[0019] Furthermore, the present invention is also a screening method for inhibitors and / or improvers of skin pigmentation symptoms, characterized by determining that a candidate substance with a reduced expression level of PSD95 in the epidermis and / or a candidate substance for which the increase in the expression level of PSD95 in the epidermis is suppressed is a better inhibitor and / or improver of skin pigmentation symptoms.

Effects of the Invention

[0020] According to the present invention, a novel screening method for an inhibitor and / or an improver of skin pigmentation symptoms can be provided.

Brief Description of the Drawings

[0021]

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Best Mode for Carrying Out the Invention

[0022] Hereinafter, preferred embodiments of the present invention will be described. However, it goes without saying that the technical scope of the present invention is not limited to the following embodiments.

[0023] The screening method of the inhibitor and / or improver for skin pigmentation symptoms of the present invention uses the degree of contact between nerve fibers and melanocytes (Mc) as an index.

[0024] In the present invention, the contact between nerve fibers and melanocytes (Mc) includes synapse-like contact. Specifically, even in a mode where there is a minute gap (synaptic cleft) between nerve fibers and melanocytes (Mc) but they are substantially in contact, the contact between nerve fibers and melanocytes (Mc) in this specification is also included.

[0025] In the present invention, the pigmentation symptom refers to a symptom in which melanocytes (Mc) become melanotic, and as a result, the color of the skin becomes dark.

[0026] Examples of the pigmentation symptom include freckles and chloasma.

[0027] Here, it is preferable that the present invention is in a form of screening an inhibitor and / or improver for freckles.

[0028] In addition, in the present invention, it is preferable to screen an inhibitor and / or improver for skin pigmentation symptoms that has an action of reducing the degree of contact between nerve fibers and melanocytes (Mc) and / or an action of suppressing the contact of nerve fibers with melanocytes (Mc).

[0029] In the screening method of the present invention, it is preferable to determine that a candidate substance with a reduced degree of contact between nerve fibers and melanocytes (Mc) and / or a candidate substance in which the contact of nerve fibers with melanocytes (Mc) is confirmed to be suppressed is a better inhibitor and / or improver for skin pigmentation symptoms.

[0030] The screening method of the present invention preferably includes adding a candidate substance to a co-culture system of nerve cells and melanocytes (Mc) and measuring the degree of contact between nerve fibers and melanocytes (Mc) in the cells.

[0031] As a method for measuring the degree of contact between nerve fibers and melanocytes (Mc) in cells, methods such as measuring the ratio of contacting melanocytes (Mc) and nerve fibers per predetermined area and measuring the number of melanocytes (Mc) with hue changes per predetermined area by time-lapse photography can be preferably mentioned.

[0032] Here, for time-lapse photography, an inverted research microscope IX83 (equipment) / Olympus (video shooting device), MetaMorph Multi Dimensional Acquisition / Molecular Device (video shooting software), and MetaMorph Multi Dimensional Motion Analysis / Molecular Device (video analysis software) can be used.

[0033] In addition, as a method for measuring the degree of contact between nerve fibers and melanocytes (Mc) in cells, the co-localization of nerve fibers and melanocytes (Mc) can be measured, and based on the result of the co-localization, the degree of contact between nerve fibers and melanocytes (Mc) in the cells can be determined.

[0034] As a method for obtaining information on co-localization, a method of imaging a skin tissue subjected to a staining process, creating a 3D model of the skin tissue, and visualizing and determining the locations where nerve fibers are unevenly distributed and the locations where melanocytes (Mc) are unevenly distributed can be mentioned.

[0035] As a method for visualizing the locations where nerve fibers are unevenly distributed and the locations where melanocytes (Mc) are unevenly distributed in the skin tissue, a method of performing binarization and thinning processes using image analysis software Imaris (manufactured by ZEISS) can be mentioned.

[0036] As a method for comparative observation of the co-localization of nerve fibers and melanocytes (Mc), examples of the method include arranging the image visualizing nerve fibers and the image visualizing melanocytes (Mc) in parallel and performing comparative observation by an evaluator specializing in this field.

[0037] Also, as another method, an example of the method includes generating a collated image by superimposing the image visualizing nerve fibers and the image visualizing melanocytes (Mc), and performing comparative observation.

[0038] Here, there is no particular limitation on the method for generating the collated image. For example, a method for generating a collated image by superimposing the image visualizing nerve fibers and the image visualizing melanocytes (Mc) can be adopted by performing image orientation change processing and image enlargement / reduction processing so that the coincidence rate of the contour of the skin tissue to be observed is increased.

[0039] Also, it is preferable to visualize the co-localization of nerve fibers and melanocytes (Mc) in the collated video. Specifically, the co-localization of nerve fibers and melanocytes (Mc) in the collated video can be visualized and made displayable on a terminal by one or more methods selected from coloring of co-localized locations, watermarking of co-localized locations, cutting out of co-localized locations, and marking of co-localized locations.

[0040] In this specification, the phrase "using the degree of contact between nerve fibers and melanocytes (Mc) as an index" includes both the meaning of a decrease in the contact ratio between nerve fibers and melanocytes (Mc) and the meaning of suppression of contact between nerve fibers and melanocytes (Mc) (time delay from the start of the test until nerve fibers contact melanocytes (Mc)).

[0041] Also, as a method for measuring the degree of contact between nerve fibers and melanocytes (Mc) in cells, it is also possible to measure the expression level of PSD95 in the epidermis and, based on the result of the PSD95 expression level, determine the degree of contact between nerve fibers and melanocytes (Mc) in cells. That is, the "decrease in the degree of contact between nerve fibers and melanocytes (Mc)" can be determined based on the decrease in the expression level of PSD95 in the epidermis and the suppression of the increase in the expression level of PSD95 in the epidermis.

[0042] In the present invention, when the degree of contact between nerve fibers and melanocytes (Mc) in cells cultured by adding a candidate substance is statistically significantly smaller than the degree of contact between nerve fibers and melanocytes (Mc) in cells cultured without adding the candidate substance, it is preferable to determine the candidate substance as a candidate for an inhibitor and / or an improver of skin pigmentation symptoms.

[0043] Also, when the degree of contact between nerve fibers and melanocytes (Mc) in cells cultured by adding a candidate substance is smaller than 1 times the degree of contact between nerve fibers and melanocytes (Mc) in cells cultured without adding the candidate substance, it may be selected as a candidate for an inhibitor and / or an improver of skin pigmentation symptoms.

[0044] Here, the application of the candidate substance is preferably in a form applied to nerve fibers and / or melanocytes (Mc). In particular, it is preferable to apply the candidate substance to nerve fibers.

[0045] In the screening method of the present invention, the inhibitor and / or improver of skin pigmentation symptoms to be screened is not particularly limited, and examples thereof include commercially available compounds (including peptides), known compounds (including peptides), compound groups obtained by combinatorial chemistry techniques, natural components derived from plants and marine organisms, and candidate substances such as animal tissue extracts.

[0046] Extracts derived from animals and plants mean not only the extracts themselves derived from animals or plants, but also the fractions of the extracts, purified fractions, and the general names of the solvent-removed products of the extracts or fractions and purified products. In addition, examples of the plant-derived extract include extracts prepared from plants growing wild or cultivated, extracts prepared from crude drugs for Kampo medicine sold as raw materials, commercially available extracts, and the like.

[0047] Here, in the present invention, it is preferable to screen for an inhibitor and / or ameliorant of skin pigmentation symptoms that exhibits an action of reducing the degree of contact between nerve fibers and melanocytes (Mc) and / or an action of suppressing the contact of nerve fibers with melanocytes (Mc).

[0048] Hereinafter, a more specific method of the screening method of the present invention will be described with reference to FIGS. 1 and 2.

[0049] The screening method of the present invention uses a device 1 including a culture tank 11 having two cell placement portions (nerve cell placement portion 111, melanocyte placement portion 112) and a connecting portion 113 connecting the cell placement portions (nerve cell placement portion 111, melanocyte placement portion 112), a preparation step of placing nerve cells in one cell placement portion (nerve cell placement portion 111), placing melanocytes (Mc) in the other cell placement portion (melanocyte (Mc) placement portion 112), and applying a candidate substance to nerve fibers and / or melanocytes (Mc); a measurement step of co-culturing nerve cells and melanocytes (Mc) and measuring the degree of contact between nerve fibers and melanocytes (Mc); a determination step of determining that a candidate substance with a reduced degree of contact between nerve fibers and melanocytes (Mc) and / or a candidate substance for which suppression of the contact of nerve fibers with melanocytes (Mc) has been confirmed based on the results of the measurement step is a more excellent inhibitor and / or ameliorant of skin pigmentation symptoms; and is preferably an embodiment having the above.

[0050] Here, in the preparation step, it is preferable to apply the candidate substance to the nerve fibers entering from the nerve cell placement portion 111 into the connecting portion 113. By adopting the above configuration, screening of the candidate substance can be accurately performed.

[0051] Here, it is preferable that the measurement step is in a form including measurement of the contact ratio of melanocytes (Mc) and nerve fibers per a predetermined area and / or measurement of melanocytes (Mc) with a changed hue per a predetermined area.

[0052] In addition, as the device 1 including the culture tank 11 having two cell arrangement parts (nerve cell arrangement part 111, melanocyte arrangement part 112) and the connecting part 113 connecting the cell arrangement parts (nerve cell arrangement part 111, melanocyte arrangement part 112), XonaChips (registered trademark, manufactured by xona) can also be used (see https: / / www.funakoshi.co.jp / contents / 51938).

[0053] In addition, the present invention includes a preparation step of applying a candidate substance to nerve fibers and / or melanocytes (Mc), a measurement step of co-culturing nerve cells and melanocytes (Mc) and measuring the degree of contact between nerve fibers and melanocytes (Mc), a determination step of determining that a candidate substance with a decreased degree of contact between nerve fibers and melanocytes (Mc) and / or a candidate substance for which suppression of melanocyte (Mc) contact of nerve fibers has been confirmed based on the result of the measurement step is a more excellent inhibitor and / or improver for skin pigmentation symptoms, and it is preferable to adopt an embodiment having these steps.

[0054] Here, the preparation step can also be in a form of mixing nerve cells and melanocytes (Mc) and seeding them in the same dish.

[0055] In addition, the measurement step can also be in a form of first culturing nerve cells into a spherical shape (spheroid) and then co-culturing nerve cells and melanocytes (Mc) to measure the degree of contact between nerve fibers (axons) extending from the spherical nerve cells and melanocytes.

[0056] In addition, in the measurement step, first, nerve cells are cultured on one side of a membrane, melanocytes (Mc) are placed on the other side of the membrane, and the degree of contact between the nerve fibers (axons) that have passed through the membrane and the melanocytes is measured by co-culturing them with the melanocytes (Mc).

[0057] Here, the size of the holes in the membrane can be set with a diameter of 1 μm to 3 μm as a guide.

[0058] In addition, the present invention a preparation step of applying two or more candidate substances to two or more test systems each containing nerve fibers and melanocytes (Mc), a measurement step of co-culturing nerve cells and melanocytes (Mc) and measuring the degree of contact between the nerve fibers and the melanocytes (Mc), a determination step of determining that a candidate substance in which the degree of contact between the nerve fibers and the melanocytes (Mc) has decreased and / or a candidate substance in which suppression of the contact between the nerve fibers and the melanocytes (Mc) has been confirmed is a better inhibitor and / or improver of skin pigmentation symptoms based on the measurement results of the measurement step, may be an embodiment having.

[0059] Here, the measurement step includes measuring the co-localization of nerve cells and melanocytes (Mc), It is preferable that the determination step is a form of determining the degree of contact between the nerve fibers and the melanocytes (Mc) based on the co-localization of the nerve fibers and the melanocytes (Mc).

[0060] In addition, the measurement step includes staining each of the nerve fibers and the melanocytes (Mc), It is preferable that the determination step is a form of determining the degree of contact between the nerve fibers and the melanocytes (Mc) based on the coincidence rate of the staining positions of the nerve fibers and the melanocytes (Mc).

[0061] The coincidence rate of the staining positions of nerve fibers and melanocytes (Mc) can be determined by performing image rotation processing and image enlargement / reduction processing so that the coincidence rate of the staining result of nerve fibers and the staining result of melanocytes (Mc) at the same site is increased, and a method can be adopted to generate a collated image in which the staining result of nerve fibers and the staining result of melanocytes (Mc) at the same site are superimposed.

[0062] Here, there is no particular limitation on the method for immunostaining nerve fibers and melanocytes. As a staining method, for example, a method of staining nerve fibers using PGP9.5 and staining melanocytes using TYRP1 can be mentioned.

[0063] Further, the measurement step includes measuring the expression level of PSD95. In the determination step, it is preferable that the candidate substance with a decreased PSD95 expression level in the epidermis and / or the candidate substance with an inhibited increase in the PSD95 expression level in the epidermis is determined to be an excellent inhibitor and / or improver for skin pigmentation symptoms.

Example

[0064] Hereinafter, various test results supporting the findings underlying the present invention are shown.

[0065] <Test 1> Phenotypic changes of melanocytes (Mc) due to contact with nerves In Test 1, the phenotypic changes of melanocytes (Mc) due to contact with nerves were verified.

[0066] (1) Time-lapse imaging First, using a device 1 equipped with a culture tank 11 having two cell placement parts (nerve cell placement part 111, melanocyte placement part 112) and a connecting part 113 connecting the cell placement parts, nerve fibers and melanocytes (Mc) were co-cultured (see FIGS. 1 and 2).

[0067] By time-lapse imaging, the presence or absence of contact between melanocytes (Mc) and nerve fibers and the change in the hue of melanocytes (Mc) were observed (see FIG. 3).

[0068] Here, for time-lapse imaging, an inverted research microscope IX83 (equipment) / Olympus (video imaging device), MetaMorph Multi Dimensional Acquisition / Molecular Device (video imaging software), and MetaMorph Multi Dimensional Motion Analysis / Molecular Device (video analysis software) were used.

[0069] Then, the contact images of melanocytes and nerve cells obtained by time-lapse imaging were binarized, and contrast observation was performed by using the intensity of melanocytes as an index of blackness.

[0070] (2) Results and Discussion As a result of the observation, it was found that melanocytes (Mc) became blackened by contact with nerves (see FIGS. 3 to 5).

[0071] From the above, it was found that the contact between nerve fibers and melanocytes (Mc) promoted the blackening of melanocytes (Mc).

[0072] That is, from the results of this example, it was found that based on the degree of contact between nerve fibers and melanocytes (Mc), inhibitors and / or improvers for skin pigmentation symptoms can be screened.

[0073] <Test 2> Verification of the relationship between nerve fibers and melanocytes (Mc) inside the skin (1) Test materials and acquisition of skin sections for staining In this example, Caucasian-derived skin (obi), 80 years old, cheek, F1822666, size 5 mm × 5 mm (human skin) was used as the test material.

[0074] And in this example, the three-dimensional skin tissue at the pigmented site (three-dimensional skin tissue including the pigmented epidermis) and the three-dimensional skin tissue at the non-pigmented site (three-dimensional skin tissue including only the non-pigmented epidermis) in the test material were selected as the measurement sites.

[0075] First, the obtained three-dimensional skin tissue was immersed in Zamboni fixative (FUJIFILM Wako Pure Chemical Corporation: 263-01991) overnight under a 4°C atmosphere.

[0076] After immersion, picric acid removal treatment was performed by washing with 70% ethanol until the yellow color of the three-dimensional skin tissue disappeared.

[0077] Thereafter, the three-dimensional skin tissue was immersed in 10% sucrose / PBS solution overnight under a 4°C atmosphere. After immersion, the three-dimensional skin tissue was immersed in 20% sucrose / PBS solution overnight under a 4°C atmosphere.

[0078] Thereafter, cryopreservation treatment of the three-dimensional skin tissue was performed using OCT Compound. Thereafter, 30-μm skin sections for staining were prepared using a cryostat.

[0079] After air drying, the 30-μm sections were washed with PBS, and then washed with 0.3% Triton X-100 / PBS and PBS.

[0080] (2) Immunostaining step After washing, blocking treatment was performed for 30 minutes at room temperature using 1% NDS (Sigma: D9663-10ML) / PBS (Blocking solution).

[0081] After blocking treatment, reaction was carried out overnight at 4°C using the primary antibody / Blocking solution. PGP9.5 (abcam) and TYRP1 (bioLegend) were used as the primary antibodies.

[0082] After washing with PBS, reaction was carried out for 60 minutes at room temperature using the secondary antibody / Blocking solution. For the secondary antibodies, Donkey anti-rabbit IgG Alexa Flour Plus 647 (Invitrogen) and Donkey anti-mouse IgG Alexa Fluor Plus 555 (Invitrogen) were used.

[0083] After washing with PBS, mounting was performed using Prolong® Gold Antifade Mountant with DAPI (Invitrogen: P36931).

[0084] Regarding 30-μm sections of three-dimensional skin tissue at the scar site (three-dimensional skin tissue including the scarred epidermis) and 30-μm sections of three-dimensional skin tissue at the non-scar site (three-dimensional skin tissue including only the non-scarred epidermis), differences in the co-localization of nerve fibers and melanocytes (Mc) were observed by contrast by an evaluator specialized in this field (Figure 6).

[0085] In addition, by subjecting them to binarization and thinning processes using the image analysis software Imaris (manufactured by ZEISS), differences in the co-localization of nerve fibers and melanocytes (Mc) were observed by contrast (Figures 7 and 8).

[0086] (3) Results and Discussion As shown in Figures 7 and 8, the 30-μm sections of three-dimensional skin tissue at the scar site (three-dimensional skin tissue including the scarred epidermis) had a higher co-localization of nerve fibers and melanocytes (Mc) compared to the 30-μm sections of three-dimensional skin tissue at the non-scar site (three-dimensional skin tissue including only the non-scarred epidermis).

[0087] That is, from the results of this example, it was found that based on the co-localization of nerve fibers and melanocytes (Mc), the degree of contact between nerve fibers and melanocytes (Mc) can be determined.

[0088] <Test 3> Examination of Screening Methods In Test 3, an examination of screening methods was performed based on the above findings.

[0089] (1) Preparation Step First, the device 1 shown in FIGS. 1 and 2 was prepared.

[0090] Neurons were placed in the cell placement section (neuron placement section 111) of the prepared device 1, and melanocytes (Mc) were placed in the other cell placement section (melanocyte placement section 112). After placement, a candidate substance to be screened was applied to the nerve fibers entering from the neuron placement section 111 into the connecting section 113.

[0091] (2) Measurement step After the preparation step, the neurons and melanocytes (Mc) were co-cultured. By imaging the process of co-culture by time-lapse photography, the presence or absence of contact between melanocytes (Mc) and nerve fibers and the change in the hue of melanocytes (Mc) were observed.

[0092] (3) Judgment step Based on the measurement values in the measurement step, the usefulness of the candidate substance to be screened as an inhibitor and / or improver of skin pigmentation symptoms was evaluated. By the above method, an inhibitor and / or improver of skin pigmentation symptoms can be screened from among the candidate substances.

[0093] <Test 4-1> Verification of phenotypic changes in melanocytes (Mc) due to contact with nerve fibers Next, verification of phenotypic changes in melanocytes (Mc) due to contact with nerve fibers was performed.

[0094] (1) Test Sensory neurons induced to differentiate from iPS cells were seeded at 200,000 cells / well in a 24-well plate. After culturing the sensory neurons for 3 days, human melanocytes were seeded at 10,000 cells / well, and co-culture was performed for 2 days. After co-culture, the sensory neurons were fixed using 4% PFA, and the human melanocytes were stained by the immunostaining method (TYRP1).

[0095] After staining, bright-field images and fluorescence images of human melanocytes were obtained using an optical microscope and a fluorescence microscope. The obtained bright-field images were binarized, and phenotypic changes of melanocytes (Mc) due to contact (co-culture) with nerve fibers were observed. The results are shown in Fig. 9.

[0096] (2) Results and Discussion As shown in Fig. 9, it was found that co-culture of nerve fibers and melanocytes (Mc) increased the degree of melanization of melanocytes (Mc).

[0097] The above results indicate that the higher the concentration of spots, the more nerve fibers in the dermis. Therefore, from the results of the examples, it was found that based on the degree of contact between nerve fibers and melanocytes (Mc), inhibitors and / or improvers for skin pigmentation symptoms can be screened.

[0098] <Test 4-2> Verification of phenotypic changes of melanocytes (Mc) due to contact with nerve fibers Next, verification of phenotypic changes of melanocytes (Mc) due to contact with nerve fibers was carried out.

[0099] (1) Test Sensory neurons induced to differentiate from iPS cells were seeded at 200,000 cells / well in a 24-well plate. After culturing the sensory neurons for 3 days, human melanocytes were seeded at 10,000 cells / well, and co-culture was carried out for 2 days.

[0100] After co-culture, the cells were fixed with 4% PFA, and nerve fibers and melanocytes (Mc) were stained by immunostaining (nerve fibers: PGP9.5, Mc: TYRP1). After staining, bright-field images and fluorescence images were obtained using an optical microscope and a fluorescence microscope.

[0101] Based on the obtained fluorescence images, the presence or absence of contact between nerve fibers and melanocytes (Mc) was subjected to visual evaluation by experts. Also, for the binarized contact images (bright field) of nerve fibers and melanocytes (Mc), experts visually evaluated the degree of blackness of melanocytes (Mc). The results are shown below.

[0102]

Table 1

[0103] (2) Results and Discussion As shown in Table 1 and Figure 10, it was found that the degree of blackening of melanocytes (Mc) increased due to the contact between nerve fibers and melanocytes (Mc). Also, as shown in Figure 10, it was found that the expression level of TYRP1 increased due to the contact between nerve fibers and melanocytes (Mc).

[0104] From the results of the examples, it was found that based on the degree of contact between nerve fibers and melanocytes (Mc), inhibitors and / or improvers for skin pigmentation symptoms can be screened.

[0105] <Test 5> Verification of the contact frequency between nerves and melanocytes (Mc) at the freckle site Next, the contact frequency between nerves and melanocytes (Mc) at the freckle site was verified.

[0106] (1) Acquisition of test materials and measurement sites In this example, the human skin shown in Table 1 above was used as the test material. And in this example, the three-dimensional skin tissue at the freckle site (three-dimensional skin tissue including the freckled epidermis and dermis) and the three-dimensional skin tissue at the non-freckle site near the freckle site (three-dimensional skin tissue including the non-freckled epidermis and dermis) in the test material were selected as the measurement sites. Then, the three-dimensional skin tissue of the selected sites was obtained by cutting it out to the size shown in Table 1 (see Figure 1).

[0107] (2) Staining In the same manner as in Example 1, the three-dimensional skin tissue at the freckle site (three-dimensional skin tissue including the freckled epidermis and dermis: SL in Fig. 11) and the three-dimensional skin tissue at the non-freckle site (three-dimensional skin tissue including the non-freckled epidermis and dermis: non-SL in Fig. 11) were cleared and stained.

[0108] (3) Analysis The 3D image captured with 3D image analysis software (Imaris) was opened, and the Coloc function was used to detect the sites where the signals of nerves and Mc co-localized in the 3D space. Then, the ratio of the detected co-localized sites was quantified. The results are shown in Fig. 11.

[0109] (4) Results and Discussion As shown in Fig. 11, it was confirmed that the contact frequency between nerves and melanocytes was significantly higher at the freckle site (SL in the figure) than at the non-freckle site (non-SL in the figure).

[0110] <Test 6> Identification of the contact pattern between nerves and melanocytes in the skin (confirmation of the expression of synapse-related factors) Next, the contact pattern between nerves and melanocytes in the skin was identified.

[0111] (1) Test 10-μm sections were prepared from OCT-embedded normal skin tissue using a cryostat. The prepared normal skin tissue sections were washed with PBS (10 min x 3).

[0112] After washing, the normal skin tissue sections were left standing for 1 hr at RT in 1% Donkey serum / PBS (Blocking solution), and reacted at 10 / N (4 °C) with 1st Ab (synapse-related factor: PSD95, melanocyte: TYRP1) / Blocking solution. After the reaction, the sections were washed with PBS (10 min x 3) and left standing for 1 hr at RT in 2nd Ab / Blocking solution. After standing, it was washed with PBS (10 min x 3), sealed with a mounting medium containing DAPI, and observed under a fluorescence microscope. The results are shown in Fig. 12.

[0113] (2) Results and Discussion As shown in Fig. 12, the expression of PSD95 was confirmed at the contact sites between nerves and melanocytes. Here, PSD95 (postsynaptic density protein 95) is a synapse-related factor and a gene that is specifically expressed on the recipient side of the synapse (postsynaptic membrane) (M. Hara, M. Toyoda, M. Yaar, J. Bhawan, E. M. Avila, I. R. Penner, B. A. Gilchrest, J Exp Med, Oct 1;184(4):1385-95 (1996)). That is, from the results of this study, it can be said that at the contact sites between nerves and melanocytes, synapse-like contacts are made, and signal transmission is carried out from nerves to melanocytes by the secretion of neurotransmitters.

[0114] Also, from the results of this study, it was found that the more the candidate substances that decreased the PSD95 expression level in the epidermis and / or the candidate substances that suppressed the increase in the PSD95 expression level in the epidermis, the better they can be determined as inhibitors and / or improvers for skin pigmentation symptoms.

Industrial Applicability

[0115] The present invention can be applied to a method for screening inhibitors and / or improvers for skin pigmentation symptoms.

Explanation of Signs

[0116] 1 Device 11 Culture tank 111 Nerve cell placement part 112 Melanocyte placement part 113 Connecting part 114 Slide glass

Claims

1. Using the degree of contact between nerve fibers and melanocytes (Mc) as an indicator, the more a candidate substance shows a decrease in the degree of contact between nerve fibers and melanocytes (Mc) and / or a confirmed inhibitory effect on the contact between nerve fibers and melanocytes (Mc), the more it is determined to be an excellent inhibitor and / or improver for skin pigmentation symptoms, wherein the degree of contact is the ratio of melanocytes (Mc) in contact with nerve fibers per a predetermined area, the co-localization of nerve fibers and melanocytes (Mc), or the number of melanocytes (Mc) with a hue change per a predetermined area, A screening method for an inhibitor and / or improver for skin pigmentation symptoms.

2. Measuring the co-localization of nerve fibers and melanocytes (Mc) and, based on the result of the co-localization, determining the degree of contact between nerve fibers and melanocytes (Mc) in cells, Regarding the information on co-localization, obtained by imaging a skin tissue subjected to a staining process, creating a 3D model of the skin tissue, and visualizing and determining the locations where nerve fibers are unevenly distributed and the locations where melanocytes (Mc) are unevenly distributed, and / or obtained by generating a collated image by overlapping an image visualizing nerve fibers and an image visualizing melanocytes (Mc) and performing comparative observation, the screening method according to Claim 1.

3. As a method for measuring the degree of contact between nerve fibers and melanocytes (Mc) in cells, measuring the expression level of PSD95 in the epidermis and, based on the result of the PSD95 expression level, determining the degree of contact between nerve fibers and melanocytes (Mc) in cells, the screening method according to Claim 1 or 2.

4. The more a candidate substance shows a decrease in the expression level of PSD95 in the epidermis and / or an inhibitory effect on the increase in the expression level of PSD95 in the epidermis, the more it is determined to be an excellent inhibitor and / or improver for skin pigmentation symptoms, the screening method according to any one of Claims 1 to 3.

5. The screening method according to any one of Claims 1 to 4, wherein the pigmentation symptom is a stain.

6. Screening an inhibitor and / or improver for skin pigmentation symptoms that has an effect of reducing the degree of contact between nerve fibers and melanocytes (Mc) and / or an inhibitory effect on the contact between nerve fibers and melanocytes (Mc), the screening method according to any one of Claims 1 to 5.

7. Using a device comprising a culture tank having two cell placement parts and a connecting part connecting the cell placement parts, A preparation step of placing nerve cells in one cell placement part, placing melanocytes (Mc) in the other cell placement part, and applying a candidate substance to nerve fibers and / or melanocytes (Mc); A measurement step of co-culturing nerve cells and melanocytes (Mc) and measuring the degree of contact between nerve fibers and melanocytes (Mc); A determination step of determining that a candidate substance with a decreased degree of contact between nerve fibers and melanocytes (Mc) and / or a candidate substance for which suppression of contact between nerve fibers and melanocytes (Mc) has been confirmed based on the results of the measurement step is a better inhibitor and / or improver of skin pigmentation symptoms; The screening method according to any one of claims 1 to 6, characterized by comprising the above steps.

8. The measurement step in the screening method according to claim 7 includes measurement of the contact ratio of melanocytes (Mc) and nerve fibers per predetermined area and / or measurement of melanocytes (Mc) with a changed hue per predetermined area.

9. A preparation step of applying a candidate substance to nerve fibers and / or melanocytes (Mc); A measurement step of co-culturing nerve cells and melanocytes (Mc) and measuring the degree of contact between nerve fibers and melanocytes (Mc); A determination step of determining that a candidate substance with a decreased degree of contact between nerve fibers and melanocytes (Mc) and / or a candidate substance for which suppression of contact between nerve fibers and melanocytes (Mc) has been confirmed based on the results of the measurement step is a better inhibitor and / or improver of skin pigmentation symptoms; The screening method according to any one of claims 1 to 6, characterized by comprising the above steps.

10. A preparation step of applying two or more candidate substances to two or more test systems including nerve fibers and melanocytes (Mc) respectively; A measurement step of co-culturing nerve cells and melanocytes (Mc) and measuring the degree of contact between nerve fibers and melanocytes (Mc); A determination step of determining that a candidate substance with a decreased degree of contact between nerve fibers and melanocytes (Mc) and / or a candidate substance for which suppression of contact between nerve fibers and melanocytes (Mc) has been confirmed based on the measurement results of the measurement step is a better inhibitor and / or improver of skin pigmentation symptoms; The screening method according to any one of claims 1 to 6, characterized by comprising the above steps.

11. The measurement step includes measuring the co-localization of nerve cells and melanocytes (Mc). The determination step is characterized in that the degree of contact between nerve fibers and melanocytes (Mc) is determined based on the co-localization of nerve fibers and melanocytes (Mc). The screening method according to claim 9 or 10.

12. The measurement step includes staining each of nerve fibers and melanocytes (Mc). The determination step is characterized in that the degree of contact between nerve fibers and melanocytes (Mc) is determined based on the coincidence rate of the staining positions of nerve fibers and melanocytes (Mc). The screening method according to claim 9 or 10.

13. The measurement step includes measuring the expression level of PSD95. The determination step is characterized in that a candidate substance with a decreased expression level of PSD95 in the epidermis and / or a candidate substance with an increased expression level of PSD95 in the epidermis inhibited from increasing is determined to be an excellent inhibitor and / or improver for skin pigmentation symptoms. The screening method according to claim 9 or 10.

14. A screening method for an inhibitor and / or improver for skin pigmentation symptoms, characterized in that a candidate substance with a decreased expression level of PSD95 in the epidermis and / or a candidate substance with an increased expression level of PSD95 in the epidermis inhibited from increasing is determined to be an excellent inhibitor and / or improver for skin pigmentation symptoms.

15. Based on the result of the expression level of PSD95, the degree of contact between nerve fibers and melanocytes (Mc) in cells is determined. A candidate substance with a decreased degree of contact between nerve fibers and melanocytes (Mc) and / or a candidate substance for which inhibition of the contact between nerve fibers and melanocytes (Mc) has been confirmed is determined to be an excellent inhibitor and / or improver for skin pigmentation symptoms. The screening method for an inhibitor and / or improver for skin pigmentation symptoms according to claim 14.

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