JP2019122266A - Screening method and kit of heel roughening suppression agent - Google Patents

Abstract

To provide a method for screening an effective substance for improving roughening unique to a heel.SOLUTION: There are provided a method for screening a substance including a heel roughening suppression effect including steps (A)-(D) and an evaluation kit using the method, the step (A) is a step in which a cultivation skin model and a test substance are made to coexist, the step (B) is a step for applying a load to the cultivation skin model, the step (C) is a step for extracting a gene or protein from the cultivation skin model, and the step (D) is a step for determining a substance having a heel roughening suppression effect with at least one gene selected from keratin 1, keratin 10 or protein expression amount and a gene or protein expression amount of involucrin, as indexes.EFFECT: As an effect, the effective substance for improving roughening unique to a heel can be screened, according to the screening method and the kit.SELECTED DRAWING: Figure 2

Description

The present invention relates to a method of screening a material having a roughening inhibitory effect and a kit therefor. Specifically, the expression level of one or more genes and / or proteins selected from keratin 1 and keratin 10 in the cultured epidermis model and the load stimulation using the expression level of involucrin gene and / or protein as an index The present invention relates to a method of screening a material having a roughening inhibitory effect and a kit therefor.

Among various troubles that occur in the sole, it is known that roughness with symptoms such as bulkiness, dusting, and cracking usually occur everyday regardless of season or gender. Not only does it impair the beauty of the appearance from a cosmetic point of view, but also causes pain and discomfort during walking when it is accompanied by pain, which may be harmful to health, and a proposal for a fundamental solution is desired. There is. However, there have been few examples that have investigated and reported in detail the mechanism by which roughening occurs and the fundamental solution is not well known.

Heretofore, as a countermeasure against roughening, a cosmetic having exfoliating horny using a peeling (patent document 1) or a grinder (patent document 2) or the like, moisturizing (patent document 3) or a horny layer softness effect (patent document 4) Although various means such as use have been proposed, these measures are aimed at alleviating the symptoms caused by roughening and are not in line with the causes of roughening, so their effects are temporary. There was also a problem that it recurred.

The skin is roughly divided into three layers, from the outside the epidermis, the dermis, and the subcutaneous tissue, but the stratum corneum is the outermost layer in the epidermis and mainly contains dead cells and water. It is composed of intercellular lipids. The stratum corneum plays a role of preventing excessive water evaporation from the body and preventing various kinds of external stimuli and infiltration of foreign matter, and such function is called a barrier function.

From the basal cells located at the lowermost part of the epidermis, the cells (keratinocytes) born by cell division are gradually pushed up, and while differentiating and maturing while producing the necessary substances, they reach up to the stratum corneum cells (keratinocytes) The process is called keratinization. In the epidermis, normality is maintained by precisely adjusting the balance of cell division and keratinization of basal cells. With keratinization, different keratin molecular species are expressed, for example, keratin 5 (K 5), keratin 14 (K 14) in basal cells, keratin 1 (K 1), keratin 10 (K 10) etc. in spiny cells. Furthermore, in the process of terminal keratinization (terminal differentiation), a marginal zone (cornified envelope) that makes the stratum corneum cell tough structure (cross-linked and insolubilized proteins such as involucrin (IVL) and loricrin by transglutaminase I. It is formed by The marginal zone contributes to the construction of a stable keratinocyte structure and is an important structure for the stratum corneum to function as a barrier. Therefore, it has been known that the degree of keratinization can be evaluated using the amounts of K5, K14, K1, K10, IVL, etc. as an index.

For example, the application of an agent for promoting keratinization to epidermal cells induces the promotion of expression of keratin 1, keratin 10, and involucrin (Non-patent document 1), conversely, the application of an agent for suppressing keratinization is keratin 1, keratin 10, It induces the suppression of expression of involucrin (Non-patent Document 2).
However, skin diseases and dry skin with rough skin on appearance may be accompanied by changes related to the degree of keratinization such as cell division, and only those whose expression levels of keratin 1, keratin 10 and involucrin are enhanced However, there are some in which keratin 1 and keratin 10 decrease and the involucrin expression area spreads (non-patent documents 3 and 4), and it is completely known about what change has occurred in these amounts in roughening. It was not done.

JP 2005-200391 A JP, 2001-62724, A Patent No. 3979497 JP, 2010-241748, A

Adeline F. Deyrieux et al. Journal of Cell Science. 2007; 120 (1): 125-136. M. Gschwandtner et al. Allergy. 2013; 68 (1): 37-47. Hideki Maejima. Clinical Derma. 2013; 15 (3): 3-6. Akemi Ishida-Yamamoto et al. J Invest Dermatol. 1995; 104 (3): 391-395.

The present invention has been made in view of the above background, and an object of the present invention is to provide a method of screening for an effective substance that improves the specific roughness of a sputum.

[1] As a first invention,
The method of screening the substance which has the roughening control effect containing the step of following (A)-(D).
(A): A step of coexistence of a cultured epidermal model and a test substance
(B): Step of applying a load to the cultured epidermal model (C): step of extracting a gene from the cultured epidermal model
(D): (d-1) and (d-2) determining the substance having a roughening inhibitory effect with the index (d-1): at least one or more genes selected from keratin 1, keratin 10 Expression level and / or protein expression level,
(D-2): gene expression amount and / or protein expression amount of involucrin [2] As a second invention, in the step (D) of the first invention,
A screening method of decreasing a gene expression level of at least one or more selected from keratin 1 and keratin 10, and judging a test substance which does not change the gene expression level of involucrin as a substance having a roughening inhibitory effect.
[3] As a third invention,
The method of screening the substance which has the roughening control effect containing the step of following (A)-(D).
(A): A step of coexistence of a cultured epidermal model and a test substance
(B): Step of applying a load to the cultured epidermis model (C): step of extracting a protein from the cultured epidermis model
(D): Determining a substance having an anti-corrosion effect based on the expression level of at least one protein selected from keratin 1 and keratin 10 and the expression level of protein of involucrin [4] as a fourth invention Is the step (D) in the third invention,
A screening method of reducing a protein expression amount of at least one or more selected from keratin 1 and keratin 10 and determining a test substance which does not change the protein expression amount of involucrin as a substance having a roughening inhibitory effect.
[5] As a fifth invention,
The method of screening the substance which has the roughening control effect containing the step of following (A)-(C).
(A): A step of coexistence of a cultured epidermal model and a test substance
(B): applying a load to the cultured epidermal model (C): preparing a tissue section from the cultured epidermal model and determining a substance having a roughening inhibitory effect by using a change in thickness of the stratum corneum as an index [6] 6 As an invention,
An evaluation kit using the method of the first to fifth inventions.

  The screening method and kit of the roughening inhibitor according to the present invention make it possible to screen for an effective substance that improves the roughening peculiar to chewing.

Photographs of normal and rough groups (typical example) Weight of normal group and rough group Softness of normal and rough group skin Epidermal stratum corneum water content of normal group and rough group Transepidermal water loss in healthy and rough groups Stratum corneum cell area of normal group and rough group Effect of weight stimulation on cell viability Changes in keratin 1 gene expression due to load stimulation Changes in keratin 10 gene expression due to load stimulation Changes in involucrin gene expression by load stimulation Change of angular layer thickness by load stimulation

  The culture epidermal model in the present invention is not particularly limited, and for example, a three-dimensional culture model in which human normal epidermal cells are layered and cultured may be used. , EPISKIN (SkinEthic) etc. can be used.

In the present invention, "to apply a load" is intended to apply a compression stimulus to the cultured epidermis model by any means. For example, there are measures such as placing a weight, pressurizing with air, compressing with water, and the like. The strength is not particularly limited. There is no particular limitation on the method of applying load stimulation to the cultured epidermis model. A load may be applied to the whole culture epidermal model, or a part may be applied. For example, a weight within a range where cell death does not occur when a load is applied can be placed on cells for stimulation. In addition, the time, interval, and number of times to give stimulation are not limited. It is known that changes in gene expression level due to load stimulation become more pronounced as the number of stimulations increases, while the cultured epidermal model gradually loses its normal stratum corneum function even in normal culture when the culture period is extended Therefore, the conditions should be selected in consideration of these changes.

In the present invention, “to be used as an indicator” means, for example, the expression amount of a gene, using the expression amount of one or more genes selected from keratin 1 and keratin 10 and the change amount of the expression amount of involucrin gene as an indicator “Change” in “amount of change” is intended to include the case where the amount of expression does not increase, decrease, or does not change. For example, a comparison may be made between the presence or absence of a test substance (test group · blank group) after subjecting to load stimulation, or one that a load stimulus was given in the presence of a test substance (test group) and no test substance · no stimulation It may be a comparison with a substance (control group), and the comparison can determine the efficacy of the test substance. In the case of using the amount of protein as an indicator, it may be considered similarly to this.
Furthermore, when using only the expression levels of keratin 1, keratin 10, and involucrin gene as an index, gene expression for keratin 1, keratin 10 is not the case where only the expression levels of keratin 1, keratin 10, and involucrin proteins are used as an index. For the amount and involucrin, when protein expression level is used as an indicator, for keratin 1 and keratin 10, protein expression level and for involucrin, gene expression level can be used as an index.

In the present invention, "the amount of expression decreases" means, for example, in the case of the amount of gene expression, when a test group (with load and with a test substance) and a blank group (with load and with no test substance) are compared. The amount of expression of the gene may be reduced. The degree of reduction may be appropriately set according to the degree of effect to be obtained, but when the test group and the blank group are compared, if there is a difference of preferably about 10%, more preferably about 25%, a more effective effect substance It can be expected to choose.
Also in the case of "the amount of protein expression decreases", it is the same meaning.

In the present invention, "no change in the expression level" does not mean that there is no mathematical change at all. It means that there is substantially no change in consideration of the general variation of the test. Specifically, for example, in the case of the gene expression level, the gene expression levels of the test group and the blank group may be compared, and the difference may not be substantially different. Considering the general variation of the test, if the difference is approximately ± 5%, it is an indication that it can be judged that there is no difference.
Also in the case of "there is no change in the expression level of protein", the same purpose is obtained.

A known method may be used as a method of extracting a gene or a protein from a cultured epidermal model. For example, in the case of gene extraction, RNeasy Mini Kit (Qiagen) or the like can be used, and the extracted total RNA may be further purified to mRNA only if necessary.

The method for confirming the gene expression level of the present invention may be a known method. It can measure using well-known methods, such as a nucleic acid hybridization method, RT-PCR method, real-time PCR method, subtraction method, and cross hybridization method using immobilized samples, such as a gene chip and an array.

  When confirming the expression level of protein, it can be measured using a known method such as Western blotting, ELISA, immunoprecipitation and the like.

  In the present invention, the change in the thickness of the stratum corneum measured using a section prepared from a cultured epidermis model can also be used as an index. Changes in the thickness of the stratum corneum may be used alone as an indicator, or may be used as an indicator together with gene expression or protein expression. By using a plurality of factors as indicators, more preferable substances can be selected.

The test substance is not particularly limited. Extracts from plants and animals, cultures of fungi or their enzyme-treated products, compounds or derivatives thereof can be used as test substances, and they may be powders, gels, etc. in addition to liquids. When it is not dissolved in the medium as it is, it can be used as a test substance by dissolving it by appropriately using a solubilizing agent such as a surfactant. Furthermore, the method of extraction is not particularly limited. With regard to the concentration to be added, any concentration may be used as long as the cells are not apparently killed 24 hours after the addition of the test substance such as the extract. In addition, the step of coexisting the test substance with the cultured epidermis model may be performed before, after, or simultaneously with applying load stimulus to the cultured epidermis model. Examples of the method for causing the test substance and the culture epidermis model to coexist include a method of adding the test substance to the culture medium side of the culture epidermis model, a method of adding the test substance to the stratum corneum side, and the like.

The kit using the screening method of the present invention is not particularly limited as long as the method is used. One kit may be equipped with the screening method of the present invention, or it may be divided into two or more kits. For example, a culture epidermal model, a medium suitable for this, a set of weights to be used, and the like can be considered.

  Hereinafter, the present invention will be more specifically described by way of examples, but the present invention is not limited by these examples.

<Test 1> Relationship between roughening and various factors The skin measurement test was carried out on the following items for 30 males and females aged 20 to 40 who obtained consent to the test. Each measurement was performed after washing the subject's feet with warm water at around 33 ± 2 ° C. and acclimating to the measurement environment (23 ± 2 ° C., 5% RH) for 10 minutes.

<Measurement item and method>
1. Body Weight The weight of each subject was measured by a body composition meter (inner scan BC-202, TANITA).
2. With the center of the buttocks of the soft soles of the skin as the measurement site, the subject is in a prone position on the bed, with the toes hanging down so that the instep is perpendicular to the top of the bed Measure the center of the buttocks several times with a hardness / elasticity measuring device (BLS 780 Ballistomer, DIA-STRON), and calculate the average value of the Indent values of three points with ak value of 0.97 to 1.03, Was the softness of the skin.
3. Epidermal stratum corneum water content The subject measured seven points at the center of the buttocks with the skin stratum corneum water content measuring device (SKICON-200EX-USB, YAYOI) in a state similar to the measurement of skin softness, and the upper limit The average value of five points excluding the lower limit value was calculated and used as the epidermal stratum corneum moisture content.
4. Transepidermal water transpiration amount The subject measures the center of the buttocks with a portable moisture transpiration meter (VapoMeter, Delfin) in the same posture as skin softness measurement, calculates the average value, and transepidermis It was the amount of water evaporation.
5. Stratum corneum cell area 5-1. The stratum corneum cells were exfoliated from the center of the buttocks of the subjects from which stratum corneum cells were collected using a horny checker (keratin checker plastic plate type, PROMOTOOL).
5-2. Hematoxylin-eosin staining of the stratum corneum cell area was collected, and then photographed with a fluorescence microscope (BZ-X710 All-in-One Fluorescence Microscope, Keyence), and the photographed image was used as image processing software (Image J) The mean value of the stratum corneum cell area present in a certain compartment was calculated and used as the stratum corneum cell area.
6. Photographed by the digital single-lens reflex camera (OLYMPUS PEN Lite E-PL6, OLYMPUS) the footpad area of the rough evaluation subject, and the criteria by which 15 evaluators visually indicate rough conditions using Table 1 in Table 1 It rated it in six steps.

7. Analysis method From the result of the rough evaluation, the normal group (levels 0 and 1) with smooth eyelid surface represented by skin condition as shown in Fig. 1 and fine cracks and dust on the surface were found, and the whole was bulky Two groups of rough groups (levels 4 and 5) showing the appearance were extracted, and the measurement results were compared and evaluated in the two groups.

<Results and Discussion>
The results are shown in FIGS. As shown in FIGS. 2-6, it was confirmed that the healthy group and the rough group have different characteristics in each measurement item.

<Weight>
The rough group in the group showed a tendency to be heavier in weight than the normal group.
Rough skin on the cheeks, back of hands, etc. is a phenomenon generally seen regardless of weight differences. It was suggested that the rough skin in the eyelid may be due to the load.

<Soft skin>
It was found that the rough skin in the rough skin is harder than the normal skin.
The water content of the stratum corneum is said to contribute to the softness of the skin, so it is possible that the skin with a small amount of water and hard and thickened can respond flexibly to mechanical irritation from the external environment, etc. Since it can not be done, it is considered to be in a weak state, and although the degree is different, it is expected that the same change occurs even in rough skin on the back of the cheek or the hand.

Transepidermal water loss and epidermal stratum corneum water content
It was confirmed that the amount of transepidermal water loss was lower and the amount of epidermal stratum corneum water content was lower in the rough group in the persimmon than in the healthy group. Generally, it is known that rough skin on the cheek and back of the hand has a high transepidermal water loss and a small amount of water in the stratum corneum, but rough skin on the eyelid has a different phenomenon than the back of the cheek or hand I understood it.

<Corneal cell area>
The stratum corneum cell area tended to be larger in the rough group in the rat than in the normal group.
Generally, it is known that if the turnover is earlier than normal, normal keratinization will not be performed and the barrier function will weaken, leading to rough skin, but the area of stratum corneum is large in the eyelids, It was found that the turnover was slow and a phenomenon different from normal skin roughening had occurred.

<Summary>
In general, it is known that transepidermal water loss is high due to a decrease in barrier function and that the stratum corneum water content is low in rough skin on the cheek or back of hands caused as a result of accelerated turnover caused by inflammation etc. However, from the above results, although the turnover is slow in the rough skin in the eyebrows and the transepidermal water loss is low, it has characteristics different from the general rough skin that the stratum corneum water content is also small all right. Moreover, in the rough group, the weight is heavy, and the skin is also hard, so it is suggested that a rough phenomenon different from that of rough skin such as the cheek or the back of the hand is occurring because the load of the weight is repeated on a daily basis.

<Test 2> Load Stimulation Experiment Using Cultured Epidermis Model Since thickening of the stratum corneum by load stimulation was considered as a factor of roughening of the eyelid skin measurement, the inventors of the present invention are about induction of stratum corneum thickness by load stimulation. The study was conducted using a cultured epidermal model.

<Materials and Methods>
1. LabCyte EPI-MODEL (Japan Tissue Engineering), which is a human three-dimensional cultured epidermal model in which human normal epidermal cells are cultured in a stratified manner, was used as a test to reproduce load stimulation to the eyelid by cultured epidermal model weight.
2. The load stimulation to a weight culture epidermal model used the weight created combining stainless steel bolts and nuts.
The weight was placed on the cells and stimulation was applied, and it was confirmed that it had no effect on the number of surviving cells when cultured, and then the weight was determined and used.
3. Load Stimulated Cultured Epidermis Model Human Three-Dimensional Cultured Epidermis Model LabCyte EPI-MODEL (Japan Tissue Engineering) is cultured at 37 ° C., 5% CO ₂ , and once every 2-3 days Assay Medium [EPI-MODEL ] (Japan Tissue Engineering) was exchanged.
As a reproduction of the load stimulation in the eyelid skin, the culture skin model was given a load stimulation, and the effect was confirmed by comparing the group given the load stimulation with the group not given the load stimulation. In the load stimulation group, weights (about 4.5 g) were placed on each skin model cup for 30 minutes, after which the weights were removed and culture was continued after replacing the medium.
The above culture was repeated five times, from the medium replacement to the next medium replacement as one culture.
The gene expression level change due to load stimulation was measured by the following experimental method 9 days after the start of culture.
4. Evaluation of changes in expression level after load stimulation The cultured epidermis model after culture confirmed that load stimulation had no significant effect on cell viability by WST-8 using Cell Counting Kit-8 (Dojin Kagaku) Figure 7).
After confirmation of cell viability, the skin model cup was washed with phosphate buffered saline (Ca, Mg free) (Phosphate Buffered Saline, PBS (-)), and the cultured epidermis model was separated using a female from the cup .
Total RNA (total RNA) in cells was separated and purified from the separated cells using RNeasy Mini Kit (Qiagen).
From total RNA isolated and purified by using a reverse transcription kit ^{PrimeScript TM RT Reagent Kit (Perfect Real} Time) ( Takara Bio) were synthesized cDNA, by reverse transcription polymerase chain reaction (Reverse Transcription Polymerase Chain Reaction, RT -PCR) The gene expression levels of several keratinization markers were analyzed quantitatively. Table 2 describes the primers used to quantify each gene expression level. The primers used were purchased from Invitrogen for synthesis.
Changes in gene expression level due to load stimulation were evaluated by comparing the group in which only culture was performed without load stimulation (without load) with the group in which load stimulation was performed (with load) (FIGS. 8 to 10). .

<Results and Discussion>
In the evaluation of the gene expression level, by applying weight stimulation to the cultured epidermis model for a fixed time, the gene expression levels of keratinization markers keratin 1 and keratin 10 are increased (Fig. 8, Fig. 9). It was confirmed that there was no increase in involucrin (FIG. 10), and that there was a tendency for gene expression different from that of a normal cultured epidermal model normally cultured without stimulation.
It is known that when cultured without stimulation, keratinization proceeds normally in the cultured epidermis model, and a stratum corneum having a sufficient barrier function is formed.
That is, it can be said that the result of the experiment indicates that the gene expression amount of the keratinization marker in the keratinization process is changed by the load stimulation, and the disturbance is generated.

5. Evaluation of stratum corneum thickening by stratum corneum layer thickness measurement After applying a load stimulus to the cultured epidermal model or culturing for a fixed period without applying a stimulus, the cultured epidermal model was separated from the skin model cup using a scalpel.
The isolated culture epidermis model is C. T. It was embedded with a compound (Sakura Finetech Japan) and it was rapidly frozen.
Frozen cells were sectioned using Cryostad (Leica Microsystems) and sectioned.
The sections were fixed using mildform (Wako Pure Chemical Industries, Ltd.).
Staining of cytoplasm and cell nucleus was performed by hematoxylin-eosin staining.
The sections after staining are photographed using a fluorescence microscope (Keyence), and the angular layer thickness is measured using free image processing software (ImageJ) using the photographed image, and the measurement results are compared to change the angular layer thickness. Was evaluated.

The results are shown in FIG.
As shown in FIG. 11, the angular layer thickness of the epidermal culture model with load was increased significantly compared to no load.

Since it was confirmed that the stratum corneum layer thickness was increased by load stimulation also in the evaluation of the stratum corneum layer thickness, it was considered that this change occurred with the change in gene expression amount of the keratinization marker.

  It is expected that in human skin of human eyelids, stimulation larger than that of weight stimulation to cells used in the above experiment is applied, and in the horny layer of eyelids, gene expression changes during the keratinization process occur more dynamically It is thought that In addition, it was predicted that the stratum corneum of the eyebrow was in a thickened state as the thickness also increased with changes in gene expression level with respect to the stratum corneum layer thickness.

From the results of the above-mentioned vaginal skin measurement test and the results of the load stimulation experiment using the cultured epidermis model, in the rough skin of the rabbit, changes in gene expression related to the keratinization process are caused by excessive load stimulation. It was thought that thickening was induced, and as a result, lack of water supply to the stratum corneum and outflow of moisturizing factors such as NMF and ceramide occurred to induce roughening.
That is, if changes in gene expression due to excessive load stimulation can be suppressed, thickening of the stratum corneum can be prevented and improved, and the induction of roughness caused by thickening of the stratum corneum can be prevented.
In other words, it can be said that the increase in gene expression levels of keratin 1 and keratin 10 caused by load stimulation can be prevented, and rough expression can be prevented by using the same expression level as in the normal state.

<Example of screening using cultured epidermal model>
1. Human three-dimensional cultured epidermal model LabCyte EPI-MODEL (Japan Tissue Engineering) is cultured at 37 ° C., 5% CO ₂ , and once every 2-3 days assay medium [EPI-MODEL] (Japan Tissue Engineering) Made a replacement.
2. In the load stimulation group, weights (about 4.5 g) were placed on each skin model cup for 30 minutes, after which the weights were removed and culture was continued after replacing the medium.
3. The test substance (commercially available plant extract) is added to the medium before the first stimulation only to give a load stimulation to a final concentration of 50 ppm or 100 ppm, and after the second time, a weight (about 4.5 g) is added to each skin model After standing for 30 minutes on the cup, after removing the weight and adding a test substance to achieve an appropriate final concentration when changing the medium, the culture was continued.
The above culture was repeated five times, from the medium replacement to the next medium replacement as one culture.
4. The change in gene expression level due to load stimulation was measured by the following experimental method 9 days after the start of culture.

<Evaluation of expression change after load stimulation>
1. The human three-dimensional cultured epidermal model after culture was confirmed by WST-8 using Cell Counting Kit-8 (Dojin Kagaku) that load stimulation did not significantly affect cell viability.
2. After confirmation of cell viability, the skin model cup is washed with phosphate buffered saline (Ca, Mg free) (Phosphate Buffered Saline, PBS (-)), and a human three-dimensional cultured epidermal model using a female from the cup Separated.
3. Total RNA (total RNA) in cells was separated and purified from the separated cells using RNeasy Mini Kit (Qiagen).
4. Reverse transcription kit from total RNA isolated and purified ^{PrimeScript TM RT Reagent Kit (Perfect Real} Time) by utilizing (Takara Bio) were synthesized cDNA, reverse transcription polymerase chain reaction (Reverse Transcription Polymerase Chain Reaction, Q -RT-PCR The gene expression levels of several keratinization markers were analyzed quantitatively by The primers described in Table 1 were used to quantify each gene expression level. The primers used were purchased from Invitrogen for synthesis.
5. By comparing the group (test group) to which the test substance was added to the culture medium (test group) and the group (blank group) to which the test substance was not added, and also comparing with the group without culture and only culture without test substance (control group), The roughening inhibitory effect of the test substance was evaluated. In order to select a substance having a higher effect, the expression level of at least one or more genes selected from keratin 1 or keratin 10 is reduced by 10% or more when the test group and the blank group are compared, and The amount of gene expression was determined to be a substance having a roughening inhibitory effect on the basis of a test substance whose change was limited to about ± 5%.

According to the present invention, it is expected that a substance having an effect of suppressing rough skin peculiar to eyebrows can be selected, and an effective substance capable of fundamentally solving rough skin in eyebrows can be provided.

Claims (6)

The method of screening the substance which has the roughening control effect containing the step of following (A)-(D).
(A): A step of coexistence of a cultured epidermal model and a test substance
(B): applying a load to the cultured epidermal model (C): extracting a gene or protein from the cultured epidermal model
(D): (d-1) and (d-2) determining the substance having a roughening inhibitory effect with the index (d-1): at least one or more genes selected from keratin 1, keratin 10 Expression level and / or protein expression level,
(D-2): gene expression amount and / or protein expression amount of involucrin (D) In the step, a test substance which decreases the expression level of at least one or more genes selected from keratin 1 and keratin 10 and does not change the expression level of involucrin is determined as a substance having a roughening inhibitory effect. The screening method of 1. The method of screening the substance which has the roughening control effect containing the step of following (A)-(D).
(A): A step of coexistence of a cultured epidermal model and a test substance
(B): Step of applying a load to the cultured epidermis model (C): step of extracting a protein from the cultured epidermis model
(D): a step of determining a substance having a roughening inhibitory effect by using the expression level of at least one or more proteins selected from keratin 1 and keratin 10 and the expression level of protein of involucrin as an index (D) In the step, a test substance which decreases the expression level of at least one protein selected from keratin 1 and keratin 10 and does not change the expression level of protein of involucrin is judged as a substance having a roughening inhibitory effect. The screening method of 3. The method of screening the substance which has the roughening control effect containing the step of following (A)-(C).
(A): A step of coexistence of a cultured epidermal model and a test substance
(B): A step of applying a load to a cultured epidermal model (C): A tissue section is prepared from the cultured epidermal model, and a step of judging a substance having a roughening inhibitory effect by using a change in stratum corneum thickness as an index An evaluation kit using the method according to any one of claims 1 to 5.