TW201619605A - Beauty care method for improving skin condition caused by reduction or increase in corneocyte desquamation, and evaluation method - Google Patents

Abstract

Provided are a beauty care method for improving a skin condition caused by the increase or reduction in corneocyte desquamation, a method for evaluating the reduction or increase in corneocyte desquamation, and a method for screening for a corneocyte desquamation promoting agent or a corneocyte desquamation suppressing agent, in each of which a novel endogenous inhibitor capable of inhibiting the activity of mesotrypsin is used. The present invention is achieved on the basis of a finding that serpin B12 can act as an endogenous trypsin inhibitor which inhibits the activity of mesotrypsin that is known as a protease involved in the starting of corneocyte desquamation.

Description

Cosmetic method and evaluation method for improving skin condition caused by inhibition or promotion of stratum corneum peeling

The present invention relates to a cosmetic method based on the close involvement of the expression of the serine protease inhibitor B12 in the stratum corneum stripping, a cosmetic method for improving the skin condition caused by the promotion/inhibition of stratum corneum peeling, and the peeling of the stratum corneum. Inhibition/promoting evaluation method, and screening method of stratum corneum exfoliation accelerator/cutin layer exfoliation inhibitor.

The outermost layer of the epidermis, the stratum corneum (SC), contains keratinized cells (keratinocytes) and a continuous extracellular lipid layer that form a physiochemical barrier to effectively protect the body from environmental hazards. The SC system is continuously formed by the final differentiation of keratinocytes. At the same time, cell detachment (peeling) is also generated to maintain the SC at a certain thickness. In normal exfoliation, each keratinocyte, the smaller agglomerate, detaches from the surface of the skin. This process is not perceptible to healthy people, but when in a certain pathology, there is an imbalance between the production and exfoliation of SC.

Lundstrom and Egelrud reported that chymotrypsin-like serine protease is present in SC and participates in exfoliation (Non-Patent Document 1). The enzyme is purified from the SC of the human foot as a stratum corneum chymotrypsin enzyme, and the cDNA encoding the stratum corneum chymotrypsin enzyme (complementary DNA) is derived from the human keratinocyte cDNA gene bank. (Non-Patent Document 2; Non-Patent Document 3). The present inventors have shown that not only chymotrypsin enzymes but also trypsin-like serine proteases are associated with the exfoliation process of SC other than the soles of the feet. Recent studies have shown that encoding 15 serine proteases (will The human tissue kallikrein gene family, which is called kallikrein 1 to 15 (KLK1 to 15), is located in chromosome 19q13.4 in the form of a cluster (Non-Patent Document 4; Non-Patent Document 5). Most of these serine proteases are widely present in human tissues and contain skin. Among members of the kallikrein family, kallikrein 7 (KLK7, hK7) and kallikrein 5 (KLK5, hK5) are involved in stratum corneum exfoliation (Non-Patent Document 6 and Non-Patent Literature) 7). Further, it has been reported that kallikrein 8 (KLK8, hK8) is an active serine protease in the human epidermis and participates in a protein decomposition cascade reaction in the skin barrier (Non-Patent Document 8).

On the other hand, three different trypsinogens are present in human pancreatic juice. These trypsinogens are called cationic trypsinogen, anionic trypsinogen, and tryptulinogen according to their isoelectric points. According to recent studies, these trypsinogens are encoded by different genes PRSS1, PRSS2, and PRSS3, respectively (Non-Patent Document 9). The PRSS1 gene product, the trypsinogen 1 and the PRSS2 gene product, the trypsinogen 2 line, is the main digestive pancreatic protease. The trypsinogen gene in the PRSS3 line, and at least two splicing variants (trypsinogens 3 and 4) were produced. All of these trypsinogens have a cut-off sequence DDDDK-I which is highly preserved and secreted by intestinal peptidase, which is derived from a variety of pancreatic trypsinogens (Light and Janska, 1989). ). Trypsinogen not only appears in the pancreas but also in other tissues, including a variety of tissue epithelial cells and human brain. It is thought that the matrix specificity and inhibitor sensitivity of mesotrypsin and the other two trypsin are different. Trypsin hardly cuts off the protein matrix. Its most characteristic properties are resistance to natural trypsin inhibitors, namely α1-trypsin inhibitor, pancreatic secretory trypsin inhibitor (Kazal type), soybean trypsin inhibitor (Kunitz type) and the like. These insights suggest that trypsin not only participates in intestinal digestion, but may also be involved in a variety of physiological responses.

The present inventors performed cDNA selection of a trypsinogen gene from a human keratinocyte cDNA gene library, and as a result, two isomers of the trypsinogen gene PRSS3 were used. Isolation, one of which is identical to trypsinogen 4 (brain trypsinogen), and the other differs only in the exon of the N-terminal region, and is named trypsinogen 5 (Non-patent literature) 10).

Both isomers have the activation sequence DDDDK-I. DDDDK-I has almost no negative residues due to the presence of negatively charged residues in the cut site. In contrast, intestinal peptidase has high specificity for the DDDDK-I sequence (Non-Patent Document 11). For example, the efficiency of enteric peptidase for bovine trypsinogen is 34,000 times that of enteric peptidase for bovine trypsin. Thus, gut peptidase is the only enzyme that physiologically converts trypsinogen to trypsin. It has also been found that intestinal peptidase is mainly present in the granular layer of the epidermis. The appearance of these trypsinogens and their activase enteropeptidase is consistent with the presence of trypsin in the terminal differentiation of keratinocytes.

Most of the phenomena are related to protein breakdown during terminal differentiation. In the outermost granulocytes, the nucleus and all organelles disappear, the molecular weight of keratin is reduced, and the filaggrin source is processed into filaggrin. In a recent study, a lymphoid-epithelial-Kazal-5 type inhibitor (LEKTI), which is a serine protease inhibitor, has been shown to be important for stratum corneum exfoliation. The mutant SPINK5 of the LEKTI (Lympho-Epithelial Kazal-Type-Inhibitor) gene was identified as a severe congenital ichthyosis-like red skin known as Netherton syndrome. Defective genes of patients with symptoms. The major stratum corneum stripping protease is severely inhibited by LEKTI (Non-Patent Document 12). SPINK5 deficient mice showed an over-promoting of epidermal protease and, in addition, over-decomposed by desmoglein 1 and showed symptoms similar to Netherton's syndrome. There is also a case where a plurality of epidermal kallikreins are involved in the exfoliation of the stratum corneum by the cleavage of desmoglein 1, which is regulated by LEKTI.

The stratum corneum exfoliation is produced by KLK acting on the cell despersome which expresses the cell's subsequent function and decomposing it. If LEKTI inhibits the action of KLK, the stratum corneum exfoliation is also inhibited. So far, the internal factors that hinder the activity of tryptase have not been known. The presence of an inhibitor.

[Previous Technical Literature] [Non-patent literature]

[Non-Patent Document 1] Arch. Dermatol. Res. 282:234-7 (1990)

[Non-Patent Document 2] J. Invest. Dermatol. 101:200-4 (1993)

[Non-Patent Document 3] Arch. Dermatol. Res. 283:108-12 (1991)

[Non-Patent Document 4] J. Biol. Chem. 275: 37397-406 (2000)

[Non-Patent Document 5] J. Biol. Chem. 382: 5-14 (2001)

[Non-Patent Document 6] Gene 254: 119-28 (2000)

[Non-Patent Document 7] J. Invest. Dermatol. 122: 1235-44 (2004)

[Non-Patent Document 8] J. Biol. Chem. 286:687-706 (2011)

[Non-Patent Document 9] Protein Pept. Lett. 12:457-64 (2005)

[Non-Patent Document 10] J. Invest. Dermatology, 130: 944-952 (2010)

[Non-Patent Document 11] Trends. Biochem. Sci. 14: 110-2 (1989)

[Non-Patent Document 12] J. Biol. Chem. 386: 1173-84 (2005)

[Non-Patent Document 13] J. Biol. Chem. 276: 49320-49330 (2001)

To date, the existence of an endogenous inhibitor that inhibits the activity of tryptase has not been known at all. An object of the present invention is to provide a cosmetic method for improving the skin condition caused by the promotion/inhibition of stratum corneum exfoliation, and a method for evaluating inhibition/promoting of stratum corneum exfoliation using a novel endogenous inhibitor which inhibits the activity of trypsin. And screening methods for stratum corneum exfoliation accelerator / stratum corneum stripping inhibitor.

The inventors have conducted diligent research and obtained the following shocking insight: the serine protease inhibitor B12 acts as a barrier to the initiation of proteases known as stratum corneum stripping. Trypsin activity acts as an endogenous trypsin inhibitor.

Therefore, this case contains the following inventions:

[1] A cosmetic method for improving the skin condition caused by the exfoliation of the stratum corneum by promoting the expression of the serine protease inhibitor B12 in the stratum corneum of the subject.

[2] The method according to 1, wherein the promotion of the expression of the above-described serine protease inhibitor B12 is promoted by a core promoter activity in a region of -1 bp to -150 bp of the promoter region of the serine protease inhibitor B12, And/or the obstruction of the silencer activity in the region of -150 bp to -600 bp of the promoter region of the serine protease inhibitor B12.

[3] A cosmetic method for improving the skin condition caused by the inhibition of stratum corneum peeling by suppressing the expression of the serine protease inhibitor B12 in the stratum corneum of the subject.

[4] The method of 3, wherein the inhibition of the expression of the above-described serine protease inhibitor B12 is inhibited by a core promoter activity in a region of -1 bp to -150 bp of the promoter region of the serine protease inhibitor B12, And/or the promotion of silencing activity in the region of -150 bp to -600 bp of the promoter region of the serine protease inhibitor B12.

[5] A method for evaluating inhibition of stratum corneum exfoliation, characterized in that the amount of the serine protease inhibitor B12 in the stratum corneum sample collected from the skin of the subject is measured, and the above-described silk amine is The increase in the amount of expression of the acid protease inhibitor B12 is an indicator of inhibition of stratum corneum stripping.

[6] The method according to 5, wherein the amount of trypsin expression in the stratum corneum sample is further measured, and the decrease in the amount of the tryptase is determined as an indicator of inhibition of stratum corneum stripping. .

[7] The method according to 5 or 6, which is characterized in that the core promoter activity in the region of -1 bp to -150 bp of the promoter region of the serine protease inhibitor B12, and/or the serine protease inhibitor B12 Silencer activity in the region of -150 bp to -600 bp in the promoter region The measurement is carried out, and the increase in the activity of the above-mentioned core promoter and/or the decrease in the activity of the above-described silencer are used as indicators for inhibition of exfoliation.

[8] The method according to any one of 5 to 7, wherein the inhibition of the above-mentioned stratum corneum peeling is caused by skin aging.

[9] The method according to any one of 5 to 7, wherein the inhibition of the above-mentioned stratum corneum peeling is caused by a skin condition selected from the group consisting of ichthyosis or sputum.

[10] A method for evaluating the promotion of stratum corneum exfoliation, characterized in that the amount of the serine protease inhibitor B12 in the stratum corneum sample collected from the skin of the subject is measured, and the above-described silk amine is The decrease in the amount of expression of the acid protease inhibitor B12 is an indicator of the promotion of stratum corneum stripping.

[11] The method according to 10, wherein the expression amount of trypsin in the stratum corneum sample is further measured, and the increase in the expression amount of the tryptase is promoted by the exfoliation of the stratum corneum. index.

[12] The method according to 10 or 11, which is characterized in that the core promoter activity in the region of -1 bp to -150 bp of the promoter region of the serine protease inhibitor B12, and/or the serine protease inhibitor B12 The silencer activity in the region of -150 bp to -600 bp in the promoter region was measured, and the decrease in the activity of the above-mentioned core promoter and/or the increase in the activity of the above-described silencer were used as indicators for promoting the exfoliation of the stratum corneum.

[13] The method according to any one of 10 to 12, wherein the promotion of the above-mentioned stratum corneum peeling is caused by a skin selected from the group consisting of sun exposure, dry skin, atopic dermatitis, psoriasis, Netherton syndrome or congenital ichthyosis-like red skin. Symptoms of skin condition or skin condition.

[14] A method for screening a stratum corneum exfoliation promoter, characterized in that, when a candidate drug is applied to a cultured cell, an agent that inhibits the expression of a serine protease inhibitor B12 in the cell is selected as a stratum corneum stripping agent. Promoter.

[15] The method according to 14, wherein the agent for promoting the expression of trypsin in the cell is further selected as a keratolytic agent.

[16] The method according to 14 or 15, which is characterized in that an agent which inhibits core promoter activity in a region of -1 bp to -150 bp which blocks the promoter region of the tyrosine protease inhibitor B12, and/or promotes serine The agent for silencing activity in the region of -150 bp to -600 bp of the protease inhibitor B12 promoter region is used as a keratin exfoliation promoter.

[17] The method according to any one of 14 to 16, wherein the keratin exfoliation promoting agent is used for prevention or improvement of skin aging.

[18] The method according to any one of 14 to 16, wherein the keratin exfoliation promoting agent is for the prevention or treatment of a skin condition selected from the group consisting of ichthyosis or sputum.

[19] A method for screening a stratum corneum exfoliation inhibitor, characterized in that, when a candidate drug is applied to a cultured cell, an agent which promotes the expression of a serine protease inhibitor B12 in the cell is selected as a stratum corneum stripping Inhibitor.

[20] The method according to 19, characterized in that the agent which inhibits the expression of trypsin in the cells is further selected as a stratum corneum stripping inhibitor.

[21] A method according to 19 or 20, which is characterized in that an agent which promotes core promoter activity in a region of -1 bp to -150 bp in which a promoter region of a serine protease inhibitor B12 is promoted, and/or an obstruction of serine The agent for silencer activity in the region of -150 bp to -600 bp of the protease inhibitor B12 promoter region is used as a stratum corneum stripping inhibitor.

[22] The method according to any one of 19 to 21, wherein the keratolytic inhibitor is selected from the group consisting of sun exposure, dry skin, atopic dermatitis, psoriasis, Netherton syndrome or congenital ichthyosis Prevention, improvement or treatment of skin condition or skin condition of skin disease.

[23] The method according to any one of 14 to 21, wherein the cultured cells are epidermal keratinocytes.

[24] A medical method for treating a skin condition caused by inhibition of stratum corneum exfoliation by promoting expression of a serine protease inhibitor B12 in a stratum corneum of a subject.

[25] A medical method for suppressing silk in a stratum corneum of a subject The performance of the amino acid protease inhibitor B12 treats a skin condition caused by the exfoliation of the stratum corneum.

[26] A pharmaceutical composition comprising a silk fibroin inhibitor B12 expression enhancer and for treating a skin condition caused by inhibition of stratum corneum exfoliation.

[27] A pharmaceutical composition comprising a performance inhibitor of a serine protease inhibitor B12 and for treating a skin condition caused by exfoliation of the stratum corneum.

According to the present invention, it is possible to provide a novel and useful cosmetic method for improving the skin condition caused by the promotion/inhibition of stratum corneum exfoliation, a method for evaluating inhibition/promoting of stratum corneum exfoliation, and a stratum corneum exfoliation accelerator/stratum stratum Screening method for stripping inhibitors. Thereby, the peeling of the stratum corneum can be controlled, thereby contributing to the prevention, improvement or treatment of the skin condition or skin condition caused by the inhibition/promoting of the stratum corneum peeling.

Figure 1 shows the stratum corneum stripping mechanism based on the performance of the serine protease inhibitor B12. The novel trypsin inhibitor, the serine protease inhibitor B12, blocks the activity of trypsin as a starting protease for stratum corneum exfoliation and KLK5/7/8 as a stratum corneum exfoliant, and participates in stratum corneum exfoliation.

Figure 2 is a graph showing the correlation between the serine extractant B12 (absorbance) of the stratum corneum extract and the inhibition of trypsin activity.

Figure 3 is a graph showing the inhibition of the activity of the serine protease inhibitor B12 against KLK8.

Figure 4 is a graph showing the inhibition of the activity of the serine protease inhibitor B12 against KLK5/7.

Fig. 5 is a photograph showing the tissue staining of trypsin and serine protease inhibitor B12 in skin tissue (eyelid, abdomen) having a thin stratum corneum and skin tissue (heel) having a thick stratum corneum.

Figure 6 shows differentiation of keratinocytes by serine protease inhibitor B12 (keratinization) A photo of the impact.

Fig. 7 is a photograph showing the effect of the amount of the serine protease inhibitor B12 on the differentiation (keratinization) of keratinocytes.

Fig. 8 is a photograph showing the tissue staining of the expression of trypsin in the lesion collected from a patient with atopic dermatitis.

Fig. 9 is a graph showing the activity of promoting the activity of the serine protease inhibitor B12 of each drug.

Fig. 10 is a graph showing the inhibitory activity of the serine protease inhibitor B12 of each drug.

Figure 11 shows the regions in the promoter region of the serine protease inhibitor B12 (-1500 bp (-1 bp to -1500 bp), -1200 bp (-1 bp to -1200 bp), -900 bp (-1 bp to -900 bp), - A graph of transcriptional activity in 600 bp (-1 bp to -600 bp), -300 bp (-1 bp to -300 bp), and -150 bp (-1 bp to -150 bp).

Fig. 12 is a graph showing the transcriptional activity caused by the deletion of the region of -1 bp to -150 bp in the promoter region of the serine protease inhibitor B12.

Figure 13 is a graph showing the transcriptional activity caused by the deletion of the region of -300 bp to -600 bp in the promoter region of the serine protease inhibitor B12.

The serine protease inhibitor B12 was discovered by high throughput genomic sequence and is known as a trypsin inhibitor belonging to the superfamily of serine protease inhibitors. Reported the presence of the serine protease inhibitor B12 in various organs such as brain, osteophytes, lymph nodes, heart, lung, liver, pancreas, testis, ovary or intestine, impeding the activity of trypsin or plasmin (non-patent) Document 13). However, the specific role of the serine protease inhibitor B12 in vivo has not been known until now.

The present inventors studied the process of trypsin involvement in stratum corneum stripping in the epidermis The surprising insight into the function of the serine protease inhibitor B12 as an endogenous inhibitor of mesotrypsin was obtained. The trypsin has an isoelectric point between the cationic trypsinogen and the anionic trypsinogen. Unlike these two trypsin, the trypsin has low decomposability to the high molecular protein, so it is considered that there is no controllable so far. An endogenous inhibitor of mesotrypsin. Further, the present inventors have found that the serine protease inhibitor B12 binds to KLK5/7/8 which is a keratinolytic enzyme to inhibit the activities. The novel stratum corneum peeling mechanism discovered this time is shown in Fig. 1. As can be seen from Fig. 1, when the expression of the serine protease inhibitor B12 in the stratum corneum is promoted, the activity of mesotrypsin and KLK5/7/8 is inhibited, so that the stratum corneum exfoliation is suppressed. On the contrary, if the expression of the serine protease inhibitor B12 in the stratum corneum is inhibited, the activity of mesotrypsin and KLK5/7/8 is not inhibited, so that the stratum corneum exfoliation is promoted.

Therefore, in one aspect of the present invention, there is provided a cosmetic method for improving the skin condition caused by the exfoliation of the stratum corneum by promoting the expression of the serine protease inhibitor B12 in the stratum corneum of the subject, And a cosmetic method for improving the skin condition caused by inhibition of stratum corneum peeling by suppressing the expression of the serine protease inhibitor B12 in the stratum corneum of the subject.

The skin condition which is caused by the peeling of the stratum corneum is, for example, a skin which causes acute inflammation such as sun exposure or rough skin. The skin condition which is caused by the inhibition of the peeling of the stratum corneum is, for example, skin aging caused by chronic inflammation caused by environmental changes such as aging, ultraviolet rays or dryness.

Similarly, the present invention provides a medical method for treating a skin condition caused by exfoliation of the stratum corneum by promoting the expression of a serine protease inhibitor B12 in the stratum corneum of a subject, and A medical method for inhibiting the skin condition caused by inhibition of stratum corneum peeling by inhibiting the expression of the serine protease inhibitor B12 in the stratum corneum of the subject.

The skin disease caused by the exfoliation of the stratum corneum, for example, atopic Dermatitis, psoriasis, Netheren syndrome, and congenital ichthyosis-like erythroderma, the skin diseases caused by the inhibition of stratum corneum exfoliation, for example, ichthyosis or sputum.

Further, the inventors of the present invention have the upstream region of the transcription start point (-1 bp) of the serine protease inhibitor B12 gene (NM_080474.2) [refer to the National Center for Biotechnology Information (NCBI)] ~-1500bp) is expected to be a promoter region, and the expression control region in the region was studied. It was found that there was a core promoter in the region of -1bp to -150bp, and there was silence in the region of -150bp to -600bp. The sub-, especially the region from -150 bp to -300 bp exerts a strong silencer function. Therefore, the core promoter activity in the region of -1 bp to -150 bp in the promoter region of the serine protease inhibitor B12 can be promoted, and/or -150 bp to the promoter region of the B12 promoter of the serine protease inhibitor. The performance of the serine protease inhibitor B12 is promoted by the inhibition of the silencer activity in the region of 600 bp, especially in the region of -150 bp to -300 bp. On the other hand, it is possible to block the core promoter activity in the region of -1 bp to -150 bp in the promoter region of the B1 promoter of the serine, and/or -150 bp in the promoter region of the B12 promoter of the serine protease inhibitor. The expression of the serine protease inhibitor B12 is inhibited by the activity of the silencer in the region of ~-600 bp, especially in the region of -150 bp to -300 bp. In the present case, the term "core promoter activity" means an activity for controlling the transcription initiation of a gene, and the term "silencer activity" means an activity for inhibiting transcription of a gene. These findings relating to the expression control region of the human serine protease inhibitor B12 contribute to the improvement of the skin condition caused by the inhibition/promoting of the stratum corneum peeling or the skin disease caused by the inhibition/promoting of the stratum corneum peeling. The application of the treatment.

According to another aspect of the present invention, there is provided a method for evaluating inhibition of keratinous layer peeling and a method for evaluating promotion of keratinous layer peeling, wherein the method for evaluating inhibition of keratinous layer peeling is characterized in that it is applied to the skin of a subject The amount of the serine protease inhibitor B12 in the stratum corneum sample collected was measured, and the increase in the expression amount of the above-mentioned serine protease inhibitor B12 was set as an index of inhibition of stratum corneum exfoliation; the stratum corneum Stripped The evaluation method for promotion is characterized in that the expression amount of the serine protease inhibitor B12 in the stratum corneum sample collected from the skin of the subject is measured, and the expression amount of the above-mentioned serine protease inhibitor B12 is measured. The reduction is an indicator of the promotion of stratum corneum stripping.

The collection of the stratum corneum sample can be carried out by any method, for example, the keratin of the heel or the like can be obtained by using a file or the like and collected as a stratum corneum powder. Further, from the viewpoint of simplicity, it is also possible to collect by tape peeling. The tape peeling refers to a method of collecting a stratum corneum sample by attaching a tape piece to a skin surface layer and peeling off, and attaching the skin stratum corneum to the peeled tape. When the tape peeling method is used, the performance of the serine protease inhibitor B12 can be measured by using only one piece of tape to collect the stratum corneum, and the evaluation of the stratum corneum hypertrophy using the serine protease inhibitor B12 as an index can be achieved. The preferred method of peeling off the tape is carried out by first purifying the surface layer of the skin with, for example, ethanol to remove sebum, stains, etc., and gently placing the tape piece cut into an appropriate size (for example, 2 × 5 cm). On the surface of the skin, an equal force is applied to the entire tape and flattened, and then the tape is peeled off with equal force. The tape may be a commercially available celluloid tape or the like, and for example, Scotch Superstrength Mailing Tape (manufactured by 3M Company), Seychelles (Sellotape (registered trademark); Mitsubishi Co., Ltd.), or the like can be used. The serine protease inhibitor B12 attached to the stratum corneum sample of the tape can be immersed in a suitable extract such as Tris-buffer (pH 8.0) (0.1 M Tris-HCl, 0.14 M). In the NaCl, 0.1% Tween-20), the stratum corneum was extracted and isolated from the tape and extracted.

In a preferred aspect of the invention, with respect to the expression of the serine protease inhibitor B12, it is preferred to utilize an antibody specific for the serine protease inhibitor B12 and to utilize a method known in the art, for example, to utilize a fluorescent substance. Various methods such as immunostaining methods such as pigments and enzymes, Western blotting methods, and immunoassay methods such as ELISA (Enzyme Linked Immunosorbent Assay) and RIA (Radioimmunoassay) . The antibody specific for the serine protease inhibitor B12 used in the ELISA may be a single antibody or a plurality of antibodies. Monoclonal antibody Methods for making polyclonal antibodies are well known to the practitioner, for example, in Lunstrum et al., J Biol. Chem. 1986, 261:9042-9048; Hurle et al. J Cell Science 1994, 107: 2623-2634. In the method of the present invention, a sandwich immunoassay is particularly preferred.

Alternatively, RNA (Ribonucleic Acid, ribonucleic acid) may be extracted from a sample of the stratum corneum, and the amount of mRNA (messenger RNA) encoding the serine protease inhibitor B12 may be determined. The extraction of mRNA and the determination of its amount are also well known in the art. For example, the quantification of RNA is carried out by quantitative polymerase chain reaction (PCR), for example, real-time polymerase chain reaction (RT-PCR). The selection of primers suitable for RT-PCR can be carried out by methods well known to the practitioner.

For RT-PCR analysis of the serine protease inhibitor B12 gene, for example, the following primers can be used.

Forward introduction: CTGGGTTGAATGTCAATCCC (sequence number 1)

Reverse primer: CACCGTGTTTTCATGGTCAA (sequence number 2)

In the above measurement, when the amount of the serine protease inhibitor B12 in the stratum corneum sample collected from the skin of the subject is significantly higher than that of the control value, or the amount of the tryptase is significantly In the lower case, it can be evaluated that the stratum corneum peeling is suppressed. On the other hand, when the amount of the serine protease inhibitor B12 in the stratum corneum sample collected from the skin of the subject is significantly lower than that of the control value, or the amount of trypsin is significantly higher than that of the trypsin In the case of high, it can be evaluated that the peeling of the stratum corneum is promoted. For example, the amount of the serine protease inhibitor B12 in the stratum corneum sample is 30% or more, preferably 50% or more, more preferably 70% or more, and most preferably 90% or more as compared with the control value. At the time, it can be judged that "the stratum corneum peeling is suppressed". On the other hand, for example, the amount of the serine protease inhibitor B12 in the stratum corneum sample is 30% or more lower than the control value, preferably 50% or more, more preferably 70% or more, and most preferably 90%. In the above case, it can be judged that "the stratum corneum peeling is promoted". The control value is as long as it is the silk in the stratum corneum The expression level of the amino acid protease inhibitor B12 is not particularly limited, and may be, for example, a serine protease in a stratum corneum sample in a corresponding portion collected from a healthy person having a statistically significant amount of good skin condition. The average of the amount of expression of the inhibitor B12.

In the evaluation method for inhibiting the exfoliation of the stratum corneum, the amount of trypsin expression in the stratum corneum sample can be further measured, and the decrease in the amount of expression can be used as an index for inhibiting the exfoliation of the stratum corneum. On the other hand, in the evaluation method for promoting the exfoliation of the stratum corneum, the amount of trypsin expression in the stratum corneum sample can be further measured, and the increase in the amount of expression can be used as a promotion of exfoliation. index.

In the above method for inhibiting the inhibition of stratum corneum exfoliation, the core promoter activity in the region of -1 bp to -150 bp in the promoter region of the serine protease inhibitor B12, and/or the initiation of the serine protease inhibitor B12 The silencer activity in the region of -150 bp to -600 bp in the subregion, particularly in the region of -150 bp to -300 bp, is measured, and the increase in the activity of the above core promoter and/or the decrease in the activity of the above silencer is used. An indicator of inhibition of stratum corneum stripping. On the other hand, in the evaluation method for promoting the exfoliation of the stratum corneum, the core promoter activity and/or the serine protease in the region of -1 bp to -150 bp of the promoter region of the serine protease inhibitor B12 can be The silencer activity in the region of -150 bp to -600 bp of the promoter region of the inhibitor B12, particularly in the region of -150 bp to -300 bp, is measured, and the activity of the above core promoter is decreased, and/or the above-described silencer activity is Increase the index used to promote the peeling of the stratum corneum. Methods for selecting transcriptional regulatory regions, methods for producing plastids, and methods for measuring transcriptional control activity are well known in the art. The control activity can be measured, for example, by luciferase assay, and the same method as in Example 11 below can be used.

By evaluating the inhibition/promoting of the stratum corneum exfoliation of the subject, the above-mentioned skin condition caused by the inhibition or promotion of the stratum corneum peeling or the diagnosis of the skin condition caused by the inhibition/promoting of the stratum corneum peeling can be performed.

In still another aspect, the present invention provides a screening method for a horny layer peeling accelerator And a method for screening a stratum corneum exfoliation inhibitor, wherein the screening method of the stratum corneum exfoliation promoter is characterized in that, when the candidate agent is applied to the cultured cell, the performance of blocking the fibroin protease inhibitor B12 in the cell is selected. The agent is used as a stratum corneum stripping accelerator; the screening method of the stratum corneum stripping inhibitor is characterized in that, when the candidate agent is applied to the cultured cell, the expression of the serine protease inhibitor B12 in the cell is selected to be promoted. The agent acts as a stratum corneum stripping inhibitor.

The expression of the serine protease inhibitor B12 can be determined by dissolving the candidate agent in water or a medium (for example, EpiLife (registered trademark) medium) and adding it to a screening system containing the cultured cells, and then as described above. The amount of the serine protease inhibitor B12 in the cultured cells was measured by a method well known in the art. The cultured cells are skin cells, typically epidermal cells, and more preferably keratinocytes. The cultured cells may be derived from humans or may be derived from other animals such as rats, mice, rabbits and the like.

The amount of the candidate drug to be added cannot be defined as a concentration of about 1 ng/ml to about 1 mg/ml, preferably about 10 ng/ml to 100 μg/ml, more preferably about 100 ng/ml to 10 μg/ml. The addition of the candidate agent is preferably carried out in the presence of calcium chloride and/or S100A8 or S100A8/A9. The culture conditions are not particularly limited, and it is preferably carried out at 5% CO ₂ , typically at 30 to 37 ° C for 1 to 14 hours, preferably at 34 to 37 ° C for 2 to 7 hours.

In the above measurement, for example, if the expression of the serine protease inhibitor B12 in the cultured cells is inhibited by 30% or more, preferably 50% or more, more preferably 70% or more, and most preferably 90%, compared with the control value. The above can be judged as "the horny layer peeling accelerator". On the other hand, for example, the performance of the serine protease inhibitor B12 in the cultured cells is promoted by 30% or more, preferably 50% or more, more preferably 70% or more, and most preferably 90% or more as compared with the control value. , it can be judged as "the stratum corneum stripping inhibitor". The control value is not particularly limited as long as it indicates the expression level of the serine protease inhibitor B12 in the healthy stratum corneum, and for example, it can be collected from a statistically significant amount of a healthy person having a good skin condition. The average amount of the expression of the serine protease inhibitor B12 in the cultured cells in the site.

In the screening method of the above-mentioned stratum corneum stripping accelerator, a drug which promotes the expression of trypsin in the above-mentioned cells may be further selected as a stratum corneum exfoliation promoting agent, and in the screening method of the stratum corneum stripping inhibitor, An agent that inhibits the expression of trypsin in the above cells is selected as a stratum corneum stripping inhibitor.

Further, in the screening method of the above-mentioned stratum corneum stripping accelerator, an agent which inhibits core promoter activity in a region of -1 bp to -150 bp in the promoter region of the tyrosine protease inhibitor B12, and/or a promoting filament may be selected. An agent for the silencing agent activity in the region of -150 bp to -600 bp in the promoter region of the amino acid protease inhibitor B12, particularly in the region of -150 bp to -300 bp, as a stratum corneum stripping promoter, and in the stratum corneum stripping inhibitor In the screening method, an agent which promotes core promoter activity in a region of -1 bp to -150 bp in the promoter region of the tyrosine protease inhibitor B12, and/or an element which inhibits the promoter region of the serine protease inhibitor B12 may also be selected. An agent for silencing activity in a region of -150 bp to -600 bp, particularly in a region of -150 bp to -300 bp, is used as a stratum corneum stripping inhibitor.

The horny layer peeling accelerator/keratin layer peeling inhibitor obtained by the screening method of the present invention can be used for the above-mentioned skin condition which is caused by the inhibition/promoting of the stratum corneum peeling or the skin disease caused by the inhibition/promoting of the stratum corneum peeling. Treatment.

Next, the present invention will be described in detail by way of examples. Furthermore, the invention is not limited thereby.

[Examples]

1. Taking the stratum corneum

The heel is cut by the beauty instrument WIDE SCRATCH (Sulf Number Co., Ltd.) and the product manual is used to recover the cuticle.

2. Production of cuticle extract

After measuring the weight of the recovered stratum corneum, add the lysis buffer (0.1M Tris- HCl (pH 8.0) + 0.14 M NaCl + 0.1% Tween 20), and homogenization was carried out on ice using a Tenbroeck type homogenizer (Cat. No. 357428, WHEATON) until all were in a liquid state. The protein quality was determined using a DC protein assay kit (Bio-Rad).

3. Refinement

Step 1: Desalination treatment

The refining was carried out using a system of AKTAprime (GE Healthcare) under low temperature conditions (4 ° C). The desalting column used in the desalination treatment was Hiprep 26/10 Desalting (GE Healthcare) and was carried out under the conditions of the product manual. The dissolution buffer used Tris-HCl (pH 8.0).

Step 2: Anion exchange

The refining was carried out using a system of AKTAprime (GE Healthcare) under low temperature conditions (4 ° C). Purification was carried out in anion exchange using an anion exchange column Hiprep 16/10 Q XL (GE Healthcare). The operation is carried out under the conditions of the product manual. The elution buffer was Tris-HCl (pH 8.0) + NaCl, and the concentration of NaCl was eluted by performing a concentration change of 0 mM to 500 mM.

Step 3: Hydroxyapatite

The purification was carried out using a system of Pharmacia LKB‧controller LCC-501 Plus (Pharmasia) under low temperature conditions (4 ° C). The hydroxyphosphorus lime column CHT2-I (Bio-Rad) was used and carried out under the conditions of the product manual. The elution buffer was eluted using a phosphate buffer (pH 6.8) and subjected to a concentration change of 0 mM to 500 mM.

Step 4: Strong cation exchange

The purification was carried out using a system of Pharmacia LKB‧controller LCC-501 Plus (Pharmasia) under low temperature conditions (4 ° C). Mono S 5/50 GL (GE Healthcare) was used and was carried out under the conditions of the product manual. The elution buffer was eluted using a phosphate buffer (pH 6.0) + NaCl, and a concentration change of 0 mM to 1000 mM was performed.

Step 5: Difference in isoelectric point

The purification was carried out using a system of Pharmacia LKB‧controller LCC-501 Plus (Pharmasia) under low temperature conditions (4 ° C). Mono P 5/50 GL (GE Healthcare) was used and was carried out under the conditions of the product manual. The buffer was 0.025 M Bis-Tris, pH 6.3, HCl as a starting buffer, and Polybuffer 74, pH 4.0, HCl was used for dissolution.

Step 6: Gel filtration

The purification was carried out using a system of Pharmacia LKB‧controller LCC-501 Plus (Pharmasia) under low temperature conditions (4 ° C). Mono P 5/50 GL (GE Healthcare) was used and was carried out under the conditions of the product manual. The dissolution buffer was PBS (Phosphate buffer solution, phosphate buffer) (DULBECCO's phosphate buffer ED SALINE (Sigma)).

4. Determination of inhibition of trypsin activity

The buffer for the activity assay used serine protease buffer (0.1 M Tris-HCl (pH 8.0), 0.1% Tween 20). The activity assay of tryptase was carried out using recombinant-PRSS3 (Cat. No. 3714-SE-010, R & Dsystems). As an enzyme substrate, Boc-Gln-Ala-Arg-MCA (Cat. No. 3135-V), Peptide Institute) was used. The matrix reaction was quantified by measurement of 2030 Multilabel Reader ARVO X3 (Perkin Elmer).

5. Identification of novel trypsin inhibitors

The trypsin barrier component derived from the stratum corneum extract was recovered and subjected to the following test.

(1) CBB (Coomassie Brilliant Blue, Coomassie Brilliant Blue) staining

The trypsin blocking component derived from the stratum corneum extract is subjected to SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) using 5-20% e-PAGEL (Cat. No. E-T520L, ATTO). Sodium alkyl sulfate-polypropylene After gelatin gel electrophoresis), only the gel was placed in a container, and washed with MilliQ water for 5 minutes × 3 times, and CBB Stain One Super (Cat. No. 11642-31, nacalai tesque) was used. Dyeing.

(2) Western ink dot analysis

The sample obtained by refining was subjected to SDS-PAGE using 5-20% e-PAGEL (Cat. No. E-T520L, ATTO), and then transcribed to the iBlot Gel Transfer system (Invitrogen) or semi-dry method. PVDF (polyvinylidene fluoride, polyvinylidene fluoride) film. The transcription membrane was washed with PBS for 5 minutes and 2 times, and then blocked by Immune Block (Cat. No. KN001A, DS Pharma Biomedical Co., Ltd.) for about 1 hour. After blocking, the cells were washed with PBST (Phosphate buffer solution with Tween) for 10 minutes×2 times, washed with PBS for 10 minutes×1 times, and the primary antibody was added to the transcription membrane. And allowed to react at room temperature for 1 hour or at 4 ° C for one night. The antibody to the serine protease inhibitor B12 uses anti-SERPINB12 antibody (S-15) (Cat. No. sc-85145, Santa Cruz) [concentration: 1/1000], anti-SERPINB12 monoclonal antibody (H3) -1B) (Cat. No. sc-32234, Santa Cruz) [concentration: 1/1000]. After the primary antibody reaction, the cells were washed with PBST for 10 minutes×2 times, washed with PBS for 10 minutes×1 times, and the secondary antibody was added to the transcription membrane, and allowed to react at room temperature for 1 hour or The reaction was allowed to react overnight at 4 °C. After the secondary antibody reaction, the cells were washed with PBST for 10 minutes×2 times, washed with PBS for 10 minutes×1 times, and ECL Prime Western Blotting Detection Reagent (Cat. No. RPN2232, GE Healthcare) was added to On the transcription membrane, FUJI MEDICAL X-RAY FILM (Cat. No. 4741007547, Fuji Film Co., Ltd.) was printed.

(3) LC-MS/MS (Liquid Chromatography-tandem Mass Spectrometry)

(i) trypsin digestion of protein mixtures

The dialyzed protein mixture was separately suspended in 20 μl of a 0.1% RapiGest (Waters Co., Milford, MA) / 50 mM ammonium carbonate solution. After the vortex was stirred, the enzyme digestion of trypsin (TPCK trypsin) was carried out by reductive alkylation treatment with L-1-toluenesulfonylguanidino-2-phenylethylchloromethylketone. After the digestion, in order to stop the digestion reaction and to prevent the surfactant (RapiGest) from being activated, trifluoroacetic acid was added so that the final concentration became 0.5%.

(ii) Identification of proteins by liquid chromatography/electrospray ionization tandem mass analysis

Protein identification in the enzyme-digested protein mixture was performed using the reported LC/MS/MS. Briefly, the mixture was loaded into a molten cerium oxide trap column using an autosampler (SI-2 semi-microHPLC system, Shiseido Co., Ltd., Tokyo, Japan) (100 μm inner diameter × 1 cm length, Jupiter Proteo C14) , 10 μm, Phenomenex, Torrance, CA). The gradient initial buffer (0.1% formic acid, 5% acetonitrile/distilled water) was flowed to the trap column for 30 minutes for desalting, and then the two-way valve was switched, thereby making the trap column and the molten cerium oxide analysis column (100 μm). The inner diameter x 12 cm length, Jupiter Proteo C14, 4 μm, Phenomenex) is directly connected. The peptides in the enzyme digest were separated using a 90 minute organic solvent gradient (5-75% acetonitrile). The column flow rate is adjusted to 300 to 400 nl/min by adjusting the length of the split resistant capillary (50 μm inner diameter) (50 to 200 mm). The peptide eluted from the column was directly introduced into a hybrid tandem mass spectrometer (LTQ-Orbitrap, Thermo Fisher Scientific, Waltham, MA) by electroporation. The mass spectrometer operates in a parallel mode of MS/MS (Tandem Mass Spectrometry). After obtaining a high-resolution full-spectrum scanning spectrum (decomposition energy 60,000) using an electric field FT-type mass spectrometer (Orbitrap), an ion trapping mass spectrometer (LTQ) is used to automatically obtain five peaks with stronger intensity as precursor ions. MS/MS spectrum. The acquired MS/MS spectra were provided to the database search using the SEQUEST algorithm (Thermo Fisher Scientific) operating on Bioworks software, and the protein was analyzed according to the degree of coincidence with the sequence. set. The sequence library for protein identification uses a non-redundant human protein library (NCBI, 2010). In order to minimize the pseudo-identification of proteins, a strict search criteria (Sf score 0.85, peptide probability 0.001, number of top matches: 1, precursor mass tolerance: 10 ppm, minimum number of peptides to identify proteins: 1, enzyme specificity: half- is used. Tryptic or fully tryptic peptides only).

According to the above experiment, it was found that there was an extremely close correlation between the performance of the serine protease inhibitor B12 and the trypsin inhibition (Fig. 2). Furthermore, the frequency band observed in Western blot analysis is about 72-74 kD, which is inconsistent with the 46 kD of the serine protease inhibitor B12, but this situation strongly suggests that the formation of the serine protease inhibitors B12 and KLK8 (28 kD). There are complexes.

6. KLK (KLK5/7/8) activity inhibition of the serine protease inhibitor B12

For the activity assay buffer, the activity of the serine protease inhibitor B12 against each KLK was determined using a serine protease buffer (0.1 M Tris-HCl (pH 8.0), 0.1% Tween 20). The activity assay of KLK8 was performed using trypsin (PRSS3) together with purified recombinant-KLK8. Similarly, the activity assay of KLK5 was carried out using recombinant-KLK5 (Cat. No. 1108-SE, R & D systems, Inc.), and the activity assay of KLK7 was performed using purified recombinant-KLK7. As the enzyme substrate, Boc-Gln-Ala-Arg-MCA (Cat. No. 3135-V, Peptide Research Institute) was used. The matrix reaction was quantified by measurement using a 2030 Multilabel Reader ARVO X3 (Perkin Elmer).

As shown in Fig. 3 and Fig. 4, it was found that the serine protease inhibitor B12 inhibits the activity of each KLK (KLK5/7/8).

7. Performance of serine protease inhibitor B12 and trypsin in human tissues

Skin tissue was collected from human eyelids and abdomen and heel, and mouse anti-haminase inhibitor B12 antibody (Cat. No. sc-32234, Santa Cruz) was used and recognized. Trypsin rabbit anti-trypsin antibody (Cat. No. 01-19-032000, ATHENS) was immunostained. As shown in Fig. 5, trypsin was strongly expressed in the relatively thin cornea or abdomen of the stratum corneum, and on the other hand, the performance of the serine protease inhibitor B12 was hardly detected. In contrast, in the relatively thick heel of the stratum corneum, a stronger expression of the serine protease inhibitor B12 was observed in the granular layer.

8. Epidermal differentiation of serine protease inhibitor B12 in a three-dimensional skin model

Human keratinocyte first-generation cultured cells (Normal Human Epidermal Keratinocytes (NHEK)) were cultured in a 75 cm ² Petri dish, and FuGENE(R)HD Transfection Reagent (FuGENE(R)) was used to overexpress the serine protease inhibitor B12. Cat. No. E2311, Promega) Transfected pCMV-HA/Serpin B12 (2 μg/ml) and used LipofectamineTM RNAiMAX Transfection Reagent (Cat. No.13778075, invitrogen) to attenuate the serine protease inhibitor B12 Transfection was performed on si-Serpin B12 (Cat. No. SASI_Hs02_00362273, sigma). The sequence of the siRNA is as follows.

Hs_SERPINB1_2273_s: 5'rGrGrArAUrCUrCUrCrCrArArGUrCrCrCrATT

Hs_SERPINB1_2273as: 5'UrGrGrGrArCUUrGrGrArGrArGrAUUrCrCTT

After transfection, the cells were cultured for 2 days in the incubator until confluence and exposed to air. Thereafter, culture was carried out according to the standard protocol of 3D Keratinocyte Starter Kit (Cat. No. PR3D-K-50, CELLnTEC), recovered on day 14, and tissue was stained by Hematoxylin-Eosin staining. Analysis. As a standard protocol of the 3D Keratinocyte Starter Kit (Cat. No. PR3D-K-50, CELLnTEC), specifically, the attached cell insert is placed in a cell culture dish (60 mm), and the precursor cells are correctly reproduced. The various epithelial cells of the niche environment were infiltrated with Progenitor Cell Targeting (PCT, Progenitor Cell Targeting) medium CnT-Prime medium (Cat. No. CnT-PR). Thereafter, a suspension of cultured cells transfected with pCMV-HA/Serpin B12 in NHEK was added to each insert, and CnT-Prime medium was added to the cell culture dish (60 mm). The dish was placed in a CO ₂ incubator and cultured for 2 days until the cells were fused. The medium inside and outside the insert was replaced with a special medium CnT-Prime 3D Barrier medium (Cat. No. CnT-PR-3D, CELLnTEC) for three-dimensional culture of keratinocytes which can rapidly differentiate and multiply cells, and the dish was filled. Incubate overnight in a CO ₂ incubator. As an air exposure operation for multilayering the skin layer, the entire medium inside and outside the insert was removed by an aspirator, and a differentiation medium CnT-Prime 3D Barrier medium was added to the outside of the insert. Thereafter, the differentiation medium CnT-Prime 3D Barrier medium was replaced only every 3 days outside the insert.

In the case of promoting the serine protease inhibitor B12, a residual nuclear component was observed in the stratum corneum cells, and the stratum corneum was thickened. On the other hand, when the serine protease inhibitor B12 was inhibited, keratin was confirmed. The layer becomes very thin (Figure 6).

Further, the addition amount (0.5 μg/ml, 1.0 μg/ml or 2.0 μg/ml) of the transfection of the serine protease inhibitor B12 and the culture period (3 days) after the transfection are the same as above. The program investigated the concentration dependence of the serine protease inhibitor B12 in the differentiation (keratinization) of keratinocytes.

As a result, it was confirmed that the expression amount of the serine protease inhibitor B12 was significantly correlated with the keratinization of keratinocytes (Fig. 7). In particular, when 2.0 μg/ml of pCMV-HA/Serpin B12 was used, it was found that the stratum corneum layer was very thick, and the residual core component was observed in the stratum corneum, and on the other hand, the particle layer was not flattened.

9. The performance of trypsin in patients with atopic dermatitis

Skin tissue was collected from patients with atopic dermatitis and immunostained using a rabbit trypsin-resistant rabbit antitrypsin antibody (Cat. No. 01-19-032000, ATHENS). As shown in Figure 8, a portion of the strong expression of trypsin was observed in patients with atopic dermatitis.

10. Screening of modulators of serine protease inhibitor B12

Quantitative PCR was carried out using the primer set below, and the expression of the serine protease inhibitor B12 in keratinocytes before and after the addition of each agent was measured.

Forward introduction: CTGGGTTGAATGTCAATCCC (sequence number 1)

Reverse primer: CACCGTGTTTTCATGGTCAA (sequence number 2)

The obtained values were corrected and compared based on the expression amount of G3PDH (forward introduction: GGTGAAGGTCGGAGTCAACGGATTTGGTCG (SEQ ID NO: 3), reverse primer: TATTGGAACATGTAAACCATGTAGTTGAGG (SEQ ID NO: 4)).

The cDNA used was cultured in a serum-free medium to culture keratinocytes, and 1.5 mM calcium was added after the fusion. After the culture for 2 days, the following agents were added and cultured for 24 hours, followed by Isogen (Nippon Gene) treatment and usual use. Method for preparing mRNA. Specifically, 100 μl of chloroform was added and shaken up and down until chloroform was mixed with Isogen. After allowing to stand at room temperature for 2 to 3 minutes, it was centrifuged at 4 ° C and 12000 g for 15 minutes. The supernatant was transferred to a new tube and 250 μl of isopropanol was added. Shake up and down to mix and allow to stand at room temperature for 10 minutes. After centrifugation was carried out for 15 minutes at 4 ° C and 12000 g, after removing the supernatant, 1 ml of 70% ethanol was added to the precipitate, and the mixture was shaken up and down to the extent that the precipitate was peeled off. After centrifugation at 4 ° C and 12000 g for 5 minutes, the supernatant was discarded and the ethanol was completely removed. After the precipitate was dried for about 10 minutes, the precipitate was dissolved with an appropriate amount of water. 1 μg of RNA was used and reverse transcription was carried out by a usual method and supplied to PCR.

Each of the drug solutions was dissolved in DMSO so as to be 10 mg/ml, and then diluted with PBS or a medium to have a final concentration of 10 μg/ml.

The results are shown in Fig. 9 and Fig. 10.

11. Identification of the transcriptional regulatory region of human serine protease inhibitor B12

Human Genomic DNA (Cat. No. G3041) [PROMEGA] was used as a template to select a region of about 1500 bp (-1 bp to -1500 bp) upstream of the transcription initiation site of the tyrosine protease inhibitor B12 gene. As a region, polymerase chain reaction (PCR) was used and the primers of the following SEQ ID NO: 5 to 11 were used for -1500 bp (-1 bp to -1500 bp), -1200 bp (-1 bp to -1200 bp), and -900 bp (-1 bp~). -900 bp), -600 bp (-1 bp to -600 bp), -300 bp (-1 bp to -300 bp), and -150 bp (-1 bp to -150 bp) were amplified.

-1500 bp forward primer: TTTGGTACCTAGATTGGGTGGGGAAG (sequence number 5)

-1200 bp forward primer: TTTGGTACCTTCTCTCTCTCTCTCGTG (sequence number 6)

-900 bp forward primer: TTGGGTACCGGTGAGAATGTTAACAAGG (sequence number 7)

-600bp forward primer: TTTGGTACCAGGGTCTTTGCAGATGTG (sequence number 8)

-300bp forward primer: TTTGGTACCTGGGAGAATAAACTCCTGC (sequence number 9)

-150 bp forward primer: TGGGGTACCTAGTTTTCCAGTTCTTAATAGC (sequence number 10)

Shared reverse primer: GCGGCTAGCTGTAAAACTTATAACGATC (sequence number 11)

The PCR reaction used Prime STAR (R) Max DNA Polymerase (Cat. No. R045A, TAKARA). The sequence in which the restriction enzyme KpnI and NheI were cleaved was added thereto, and the fusion reaction was carried out into the firefly luciferase reporter vector pGL4.12 [luc2CP] (Cat. No. E6671, PROMEGA), and thereafter utilized. The plastid will be competent for cell transformation. Conjugation using Ligation High (Cat.No.LGK-101, TOYOBO ), using competent cells ^{One Shot (R) OmniMAX TM 2} T1R Chemically Competent E.coli (Cat.No.C8540-03, Invitrogen). Thereafter, the automorphic body refines the target body. For the purification of the plastid, QIAprep Spin Miniprep Kit (Cat. No. 27106, QIAGEN) and Endo Free Plasmid Maxi Kit (Cat. No. 12362, QIAGEN) were used. Purified plastids were used and transfected into human keratinocyte first generation cultured cells (Normal Human Epidermal Keratinocytes (NHEK)) using FuGENE(R) HD Transfection Reagent (Cat. No. E2311, Promega). After transfection, culture was carried out for 48 hours in an incubator. Thereafter, luciferase assay using a Dual-Luciferase (R) Reporter Assay System (Cat. No. E1980, Promega) was performed, whereby the transcription regulating region of the serine protease inhibitor B12 was determined. As a standard for luciferase analysis, pGL4.74 [hRluc/TK] (Cat. No. E6921, Promega) was used.

The results are shown in Fig. 11.

It is composed of -1500 bp (-1 bp to -1500 bp), -1200 bp (-1 bp to -1200 bp), -900 bp (-1 bp to -900 bp), and -600 bp (-1 bp to -600 bp). The pGL4.12 plastid in the region of -300 bp (-1 bp to -300 bp) was used as a template, and the -1 bp to -150 bp region was deleted (Δ-1 bp to -150 bp) by polymerase chain reaction (PCR). The primers of the following SEQ ID NOs: 12 to 13 were amplified to introduce site-specific variation.

-1bp~-150bp deletion forward primer: AATGACCTGCTAGCCTCGAGGATATC (sequence number 12)

-1bp~-150bp deletion with reverse primer: AGGCTAGCAGGTCATTATCATCCCTG (sequence number 13)

Site-specific variation was introduced into the polymerase chain reaction using Prime STAR (R) Mutagenesis Basal Kit (Cat. No. R046A, TAKARA). Thereafter, the competent cells are used to transform the competent cells. Competent cells using the ^{One Shot (R) OmniMAX TM 2} T1R Chemically Competent E.coli (Cat.No.C8540-03, Invitrogen). Thereafter, the automorphic body refines the target plastid. For the purification of the plastid, QIAprep Spin Miniprep Kit (Cat. No. 27106, QIAGEN) and Endo Free Plasmid Maxi Kit (Cat. No. 12362, QIAGEN) were used. Purified plastids were used and transfected into human keratinocyte first generation cultured cells (Normal Human Epidermal Keratinocytes (NHEK)) using FuGENE(R) HD Transfection Reagent (Cat. No. E2311, Promega). After transfection, culture was carried out for 48 hours in an incubator. Thereafter, luciferase assay using a Dual-Luciferase (R) Reporter Assay System (Cat. No. E1980, Promega) was performed, whereby the transcription regulating region of the serine protease inhibitor B12 was determined. As a standard for luciferase analysis, pGL4.74 [hRluc/TK] (Cat. No. E6921, Promega) was used.

As shown in Fig. 12, almost no transcriptional activity was observed in the region of -1 bp to -150 bp, and it was found that the -1 bp to -150 bp region functions as a core promoter.

A pGL4.12 plastid comprising a region of -1500 bp (-1 bp to -1500 bp), -1200 bp (-1 bp to -1200 bp), and -900 bp (-1 bp to -900 bp) was prepared as a template for identifying a transcriptional regulatory region. In order to delete the -300 bp to -600 bp region (Δ-300 bp to -600 bp), site-specific variation introduction was carried out by polymerase chain reaction (PCR) using primers of the following SEQ ID NOs: 14 to 15.

-300bp~-600bp deletion forward primer: TGGAAATTGGGAGAATAAACTCCTG (sequence number 14)

-300bp~-600bp deletion with reverse primer: TCTCCCAATTCCAAACAAGGTCAT (sequence number 15)

Site-specific variation was introduced into the polymerase chain reaction using Prime STAR (R) Mutagenesis Basal Kit (Cat. No. R046A, TAKARA).

Further, the selection of the -300 bp to -600 bp region was carried out by using Human Genomic DNA (G3041) [PROMEGA] as a template, and amplification was carried out by polymerase chain reaction (PCR) using primers of the following SEQ ID NOs: 8 and 16.

-600bp forward primer: TTTGGTACCAGGGTCTTTGCAGATGTG (sequence No. 8)

-300bp reverse primer: TTTGCTAGCTGGTTCTGGGGGACAAAC (sequence number 16)

The PCR reaction used Prime STAR (R) Max DNA Polymerase (Cat. No. R045A, TAKARA). The sequence in which the restriction enzyme KpnI and NheI were cleaved was added thereto, and the fusion reaction was carried out into the firefly luciferase reporter vector pGL4.12 [luc2CP] (Cat. No. E6671, PROMEGA), and thereafter utilized. The plastid will be competent for cell transformation. Conjugation using Ligation High (Cat.No.LGK-101, TOYOBO ), using competent cells ^{One Shot (R) OmniMAX TM 2} T1R Chemically Competent E.coli (Cat.No.C8540-03, Invitrogen).

Thereafter, the automorphic body refines the target body. For the purification of the plastid, QIAprep Spin Miniprep Kit (Cat. No. 27106, QIAGEN) and Endo Free Plasmid Maxi Kit (Cat. No. 12362, QIAGEN) were used. Purified plastids were used and transfected into human keratinocyte first generation cultured cells (Normal Human Epidermal Keratinocytes (NHEK)) using FuGENE(R) HD Transfection Reagent (Cat. No. E2311, Promega). After transfection, culture was carried out for 48 hours in an incubator. Thereafter, luciferase analysis using a Dual-Luciferase (R) Reporter Assay System (Cat. No. E1980, PROMEGA) was carried out, whereby the transcription regulating region of the serine protease inhibitor B12 was determined. As a standard for luciferase analysis, pGL4.74 [hRluc/TK] (Cat. No. E6921, PROMEGA) was used.

In Fig. 13, the region of -150 bp to -600 bp is shown to function as a silencer. Among them, the region of -300 bp to -600 bp has an effect on the promoter region as a silencer region, but does not function as a silencer in a single case. On the other hand, it is considered that the region of -150 bp to -300 bp functions as a silencer even when it is alone.

<110> Japanese Business Shiseido Co., Ltd.

<120> Evaluation method of stratum corneum peeling

<130> P150636WO

<150> JP2014-217823

<151> 2014-10-24

<160> 16

<170> Patent version 3.5

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Claims (23)

A cosmetic method for improving the skin condition caused by the exfoliation of the stratum corneum by promoting the expression of the serine protease inhibitor B12 in the stratum corneum of the subject. The method of claim 1, wherein the promotion of the expression of the above-described serine protease inhibitor B12 is promoted by a core promoter activity in a region of -1 bp to -150 bp of the promoter region of the serine protease inhibitor B12, and / or caused by the inhibition of the silencer activity in the region of -150 bp to -600 bp of the promoter region of the serine protease inhibitor B12. A cosmetic method for improving the skin condition caused by inhibition of stratum corneum peeling by inhibiting the expression of the serine protease inhibitor B12 in the stratum corneum of a subject. The method of claim 3, wherein the inhibition of the expression of the above-described serine protease inhibitor B12 is inhibited by the core promoter activity in a region of -1 bp to -150 bp of the promoter region of the serine protease inhibitor B12, and / or the promotion of the silencing activity in the region of -150 bp to -600 bp of the promoter region of the serine protease inhibitor B12. A method for evaluating inhibition of stratum corneum exfoliation, characterized in that the amount of the serine protease inhibitor B12 in the stratum corneum sample collected from the skin of the subject is measured, and the above-described serine protease is inhibited The increase in the amount of expression of the agent B12 is an indicator of the inhibition of the peeling of the stratum corneum. The method of claim 5, wherein the amount of trypsin expressed in the stratum corneum sample is further measured, and the decrease in the amount of the tryptase is determined as an indicator of inhibition of stratum corneum exfoliation. The method of claim 5 or 6, wherein the promoter region of the serine protease inhibitor B12 The core promoter activity in the region of -1 bp to -150 bp in the domain, and/or the silencer activity in the region of -150 bp to -600 bp in the promoter region of the serine protease inhibitor B12, and the above-mentioned core initiation The increase in the sub-activity and/or the decrease in the above-described silencer activity is an indicator of inhibition of stratum corneum stripping. The method of any one of claims 5 to 7, wherein the inhibition of the exfoliation of the stratum corneum is caused by skin aging. The method of any one of claims 5 to 7, wherein the inhibition of the above-mentioned stratum corneum peeling is caused by a skin condition selected from the group consisting of ichthyosis or sputum. An evaluation method for promoting the exfoliation of the stratum corneum, which measures the expression amount of the serine protease inhibitor B12 in the stratum corneum sample collected from the skin of the subject, and the above-mentioned serine protease inhibitor B12 The decrease in the amount of performance is an indicator of the promotion of stratum corneum stripping. The method according to claim 10, wherein the amount of trypsin expression in the stratum corneum sample is further measured, and the increase in the amount of the above tryptase is used as an index for promoting the exfoliation of the stratum corneum. The method of claim 10 or 11, wherein the core promoter activity in the region of -1 bp to -150 bp of the promoter region of the serine protease inhibitor B12, and/or the promoter region of the serine protease inhibitor B12 The silencer activity in the region of -150 bp to -600 bp was measured, and the decrease in the activity of the above core promoter and/or the increase in the activity of the above silencer was used as an indicator for promoting the exfoliation of the stratum corneum. The method of any one of claims 10 to 12, wherein the promotion of the above-mentioned stratum corneum peeling is caused by a skin selected from the group consisting of sun exposure, dry skin, atopic dermatitis, psoriasis, Netherton syndrome or congenital ichthyosis-like erythroderma Skin condition or skin condition. A method for screening a keratinous layer peeling accelerator, which is characterized in that, when a candidate drug is applied to a cultured cell, a drug which inhibits the expression of the serine protease inhibitor B12 in the cell is selected as a keratolytic agent. The method of claim 14, wherein the agent which promotes the expression of trypsin in the above cells is further selected as a stratum corneum exfoliation promoter. The method of claim 14 or 15, wherein the agent which inhibits core promoter activity in a region of -1 bp to -150 bp which blocks the promoter region of the tyrosine protease inhibitor B12, and/or the tyrosine protease inhibitor B12 is selected. The agent for silencing activity in the region of -150 bp to -600 bp in the promoter region serves as a keratin exfoliation promoter. The method of any one of claims 14 to 16, wherein the keratin exfoliation promoting agent is used for prevention or improvement of skin aging. The method of any one of claims 14 to 16, wherein the stratum corneum exfoliation promoter is for the prevention or treatment of a skin condition selected from the group consisting of ichthyosis or sputum. A method for screening a keratolytic inhibitor is characterized in that, when a candidate drug is applied to a cultured cell, a drug which promotes the expression of the serine protease inhibitor B12 in the cell is selected as a stratum corneum release inhibitor. The method of claim 19, wherein the agent which inhibits the expression of trypsin in the cells is further selected as a stratum corneum stripping inhibitor. The method of claim 19 or 20, wherein the agent which promotes core promoter activity in a region of -1 bp to -150 bp which promotes the promoter region of the tyrosine protease inhibitor B12, and/or hinders the tyrosine protease inhibitor B12 An agent for silencer activity in a region of -150 bp to -600 bp in the promoter region is used as a keratolytic inhibitor. The method of any one of claims 19 to 21, wherein the stratum corneum stripping inhibitor is selected from the group consisting of sun exposure, dry skin, atopic dermatitis, psoriasis, Netherton syndrome or congenital ichthyosis-like red skin Prevention, improvement or treatment of a diseased skin condition or skin condition. The method of any one of claims 14 to 22, wherein the cultured cells are epidermal keratinocytes.