JP2010164404A - Wrinkle formation marker and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To search for and identify a biomarker related to the formation of wrinkles. <P>SOLUTION: This wrinkle marker includes any one of 17 kinds of proteins: Krt6, Krt16, Krt17, Pfn1, Wars, Pdia6, Prdx1, Ppia, Fubp1, FABP5, Pgm2, Ran, S100A9, Lgals7, Tuba1, Serpinb1, and Taldo1. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a marker that affects wrinkle formation and a technique for using the marker.

As a method of evaluating skin condition, elasticity and moisture content are measured, but it is only physical evaluation and grasping of the current skin, accurate skin condition grasping, and wrinkles are likely Future predictions that are likely to happen and clear solutions cannot be provided.
Commercially available pharmaceuticals and cosmetics are rarely considered for each individual, and many are uniform. Similar to the social flow of individual medical care in the future, in the field of cosmetics, it is expected that provision of individual (semi-made) cosmetics tailored to the characteristics of each individual will be required rather than a uniform response.
Patent Document 1 (Japanese Patent No. 3914244) discloses a wrinkle-improving cosmetic containing a composition for removing abnormal protein containing silybin and a silane extract and / or soybean saponin. Patent Document 2 (Japanese Patent Application Laid-Open No. 2007-302607) discloses an elastase inhibitor and / or a collagen production promoter characterized by containing an extract of Icelandic moss (Cetralia islandica) as an active ingredient, and ultraviolet rays. Cosmetics having an anti-wrinkle effect containing a blocking / absorbing agent are disclosed. Patent Document 3 (Japanese Patent Application Laid-Open No. 2006-306804) discloses a wrinkle formation inhibitor characterized by containing acteoside as an active ingredient, and a wrinkle formation inhibitor characterized by containing the wrinkle formation inhibitor. Cosmetic or food / beverage composition.

Japanese Patent No. 3914244 JP 2007-302607 A JP 2006-306804 A

  The purpose is to search and identify biomarkers related to wrinkle formation.

  The present inventor created a photoaging-induced wrinkle model mouse by irradiating hairless mice with ultraviolet rays having the same intensity as sunlight for a long period of time, and the external preparation proposed by the present applicant in Patent Document 1 etc. As a treatment, silybin, which is suppressed in skin aging in humans, was administered. The skin was collected from mice that had wrinkles suppressed by administration of silybin and mice that had wrinkles treated with ethanol as a control, and then only the epidermal site located on the outermost layer of the skin was LMD (Laser Microdissection), Or it extract | collected by the water bath method, and protein was extracted from the obtained epidermis. Next, using the experimental method that enables the search for the epidermis-specific protein that has been established, the protein obtained from the skin that has been treated with silybin to suppress wrinkles and the skin that has been treated with ethanol to form wrinkles, The variation was comprehensively analyzed using the 2D DIGE method. As a result, 17 fluctuating spots were confirmed. The protein in this spot was analyzed to determine the protein that was considered a candidate for a variable spot. With respect to these proteins, a variation verification experiment in mouse skin was performed using Western blotting, and 17 types of proteins correlated with wrinkle formation could be identified, leading to the present invention.

The gist of the present invention is as follows.
1. A wrinkle marker consisting of one of the following 17 proteins.
(G1):
(1) peroxyredoxin 1,
(2) peptidylpropyl isomerase A,
(3) far upstream element (FUSE) binding protein 1,
(12) ras-related nuclear protein
Protein (G2) selected from the group consisting of 4 types:
(4) Keratin 6A,
(5) Keratin 16,
(6) Keratin 17,
(7) Profilin 1,
(8) tryptophan-tRNA ligase,
(9) Protein disulphide-isomerase A6 precursor,
(10) fatty acid binding protein 5, epidermal,
(11) Phosphoglycerate Mutase 2
(13) S100 calcium binding protein A9 (calgranulin B)
(14) electin, galactose binding, soluble 7
(15) tubulin, alpha 1
(16) serine (or systemine) proteinase inhibitor, clade B, member 1a
(17) transdolase 1
1. A protein selected from the group consisting of 13 types 1. A method for examining the effect of a test substance on wrinkle formation on a skin by examining a change in the expression level of a protein serving as a wrinkle marker.
3. 1. The wrinkle formation factor is ultraviolet exposure. Described method

17 kinds of proteins whose expression levels fluctuate due to the formation of wrinkles were clarified.
The present invention can search for a substance that affects wrinkle formation using the skin stratum corneum obtained using a non-invasive technique. It is possible to prepare a wrinkle formation inhibitor or a cosmetic based on the unique characteristics of the individual who has the skin stratum corneum.

The figure which shows the test 1 wrinkle formation and an external preparation administration outline | summary. Graph showing wrinkle grading Electrophoresis spot reference diagram. (A) Electrophoresis diagram of an epidermis sample obtained by the LMD method, (b) Electrophoresis diagram of an epidermis sample obtained by the water bath method. The figure which shows a 2-dimensional difference gel electrophoresis analysis result. (A) Six analysis results of skin samples obtained by the LMD method are shown. (B) Eleven analysis results of the skin sample obtained by the hot water method are shown. The graph which shows the increase / decrease change of the expression level of the protein of six epidermis samples obtained by LMD method. The graph which shows the increase / decrease change of the expression level of 6 proteins in 11 epidermis samples obtained by the hot water method. The graph which shows the increase / decrease change of the expression level of 5 proteins in 11 epidermis samples obtained by the hot water method. Verification diagram by Western blotting (WB). (A) Six types of proteins obtained by the LMD method. (B) 11 types of proteins obtained by the water bath method.

  Hereinafter, embodiments of the present invention will be described in more detail.

The present invention has been made by searching for proteins that serve as markers for the formation and suppression of wrinkles and finding their use.
Photo-aging-induced wrinkle model mice were prepared by irradiating hairless mice with ultraviolet rays having the same intensity as sunlight for a long period of time, and silybin, which is a skin aging inhibitor in humans, was administered as an external preparation. Treat mice with wrinkles suppressed by administration of silybin and ethanol as a control, collect skin from mice that formed wrinkles, and then use the LMD method (Laser Microdissection) only for the epidermis located on the outermost layer of the skin Alternatively, the protein was extracted from the epidermis obtained by the hot water bath method. Only the epidermis site was used so that non-invasive sample collection was possible.
Next, 2D DIGE for proteins obtained from wrinkle-suppressed skin (Silybin treatment) and wrinkle-formed skin (Ethanol treatment) using established experimental methods that enable the search for epidermis-specific variable proteins The protein variation was comprehensively analyzed using the method. As a result, about 2000 protein spots were detected in total from the samples obtained from the LMD method, and dominant fluctuations were confirmed in 6 spots among them. In addition, about 2500 protein spots were detected in total from the samples obtained from the water bath method, and among them, the dominant fluctuation was confirmed in 14 spots. Of the 6 types of proteins that were confirmed to be varied in the sample obtained from the LMD method and 14 types of proteins that were confirmed to be varied in the sample obtained from the water bath method, 3 types of proteins were duplicated.

In the 2D DIGE method, for comparison data (for example, whether or not silybin is applied), the protein of one condition is colored red and the protein of the other condition is colored green. When the protein expression level is the same, yellow (because red and green overlap), while when one of the protein expression levels is low or high, the color is red or green (because red and green are not balanced), so the expression level is different This is a technique that can detect protein (not yellow, but red or green).
Next, the protein was identified using the mass spectrometer (LC-Q-TOF / MS) about the spot by which the fluctuation | variation was recognized. The obtained results were analyzed using a database (Mascot), and proteins considered as candidates for fluctuation spots were determined.
Next, a commercially available antibody specific to the protein considered to be a candidate for this variation spot was purchased, and a variation verification experiment on mouse skin was performed using the Western blotting method. There were 17 types of proteins examined, and in all of them, changes consistent with the results of proteome analysis were observed, and the changes could be verified in the skin of mice. In other words, we succeeded in identifying 17 proteins that correlate with wrinkle formation.

  The 17 types of proteins are known proteins, which are outlined below.

(1) peroxyredoxin 1
This protein is classified as a protein belonging to the peroxiredoxin family of antioxidant enzymes and reduces hydrogen peroxide and alkyl hydroperoxides. This protein acts as an anti-oxidant in the cell and may contribute to the antiviral activity of CD8 (+) T cells. This protein has a growth promoting effect and may play a role in cancer development or progression.

(2) peptidylpolymer isomerase A
This protein belongs to the peptidylprolyl cis / trans isomerase (PPIase) family. PPIases catalyze the cis-trans isomerization of prolineimide peptide bonds in oligopeptides and promote the formation of protein folding structures. This protein is a cyclosporin binding protein and may act in the immunosuppression mediated by cyclosporin A. This protein can interact with several HIV proteins such as p55 gag and Vpr and is known to be necessary for HIV virus particle formation.

(3) far upstream element (FUSE) binding protein 1
This protein activates the upstream transcription region of c-myc and stimulates c-myc expression. Regulation of the FUSE region by this protein occurs through single strand binding to the untranslated strand. The transcriptional activity of this protein may be regulated by alternative splicing, translation efficiency, and post-translational modification.

(4) Keratin 6A
This protein belongs to the keratin family and is expressed in keratinocytes that show hyperproliferation of the supra basal part of the skin. Expression is rapidly induced in injured keratinocytes. Six type polymorphisms have been identified in this type II cytokeratin, and this polymorphism is considered to be a result of continuous gene duplication.

(5) Keratin16
It is a cytoskeletal protein. It is a component of an intermediate filament that forms a heterotetramer with Keratin 6. It is expressed in hair follicles, nails, squamous epithelium layer of the oral cavity, basal layer of epithelium, palmar epithelium, and sweat glands, and is involved in cell proliferation and differentiation. Mutations in this gene are associated with type 1 congenital nail sclerosis, non-bullous palmar plantar keratosis, and unilateral palmar plantar plantar nevus.

(6) Keratin 17
It is a type I protein with intermediate filament chains. Keratin 17 is expressed in nail beds, hair follicles, sebaceous glands, and other epidermal appendages. Mutations in this gene cause Jackson-roller congenital nail sclerosis and multiple sebaceous cysts.

(7) Profilin1
This protein is a protein that is widely expressed in the living body, and is known as a protein that regulates the polymerization / depolymerization of actin, which is a cytoskeleton constituent protein in cells.

(8) tryptophan-tRNA ligase
This protein corresponds to aminoacyl tRNA synthetase and catalyzes aminoacylation of amino acid tRNA. Since the main role of these enzymes is the combination of three pairs of nucleotides and amino acids contained in tRNA, it is considered to be one of the proteins that first appeared in evolution. There are two tryptophanyl tRNA synthetases: a cytoplasmic type called WARS and a mitochondrial type called WARS2.

(9) Protein disulphide-isomerase A6 precursor
This protein belongs to the protein disulfide isomerase family, has two thioredoxin domains, and functions to catalyze the reorganization of ss bonds.

(10) Fatty acid binding protein 5, epidermal
This protein is a fatty acid binding protein found in epidermal cells. This protein was initially identified as a gene that is enhanced in psoriatic tissue. This protein is a cytoplasmic protein that binds small and highly conserved long chain fatty acids to other hydrophobic ligands. The role appears to include fatty acid uptake, transport, and metabolism.

(11) Phosphoglycerate Mutase 2
This protein is involved in glucose synthesis and metabolism.

(12) ras-related nuclear protein
This protein (RAN) is a low molecular weight GTP-binding protein belonging to the RAS superfamily that is essential for RNA and protein transport through the nuclear pore complex. This protein is involved in the regulation of DNA synthesis and cell cycle progression. Since RAN is multifunctional, it is thought to interact with several other proteins. RAN regulates microtubule network formation and organization. RAN may be the primary signaling molecule that coordinates microtubule polymerization during mitosis.

(13) S100 calcium binding protein A9 (calgranulin B)
This protein belongs to the S100 protein family with two EF hand calcium binding motifs. S100 protein is localized in the cytoplasm and nucleus of various cells and is involved in the regulation of many cellular processes such as cell cycle progression and differentiation. There are at least 13 genes for S100, which are concentrated on chromosome 1q21. This protein has the potential to function in the inhibition of casein kinase, and its altered expression is associated with cystic fibrosis.

(14) electin, galactose binding, soluble 7
Galectins are β-galactoside-binding proteins involved in the regulation of cell-cell interactions and cell matrix interactions. Differential hybridization indicates that this lectin is specifically expressed in keratinocytes. It is expressed at all stages of the epidermis (ie basal layer and upper basal layer) and moderately suppressed by retinoic acid. This protein was found mainly in the stratified squamous epithelium. Intracellular localization and marked decrease in expression in cultured keratinocytes indicate a role in cell-cell interactions and cell matrix interactions required for normal growth regulation.

(15) tubulin, alpha 1
It is a protein found in the cytoskeleton of eukaryotic cells and fulfills a variety of functions as mitosis, cytoskeleton transport and auxiliary structures. It is composed of heterodimers of α and β tubulin. These proteins belong to the tubulin superfamily and are composed of six different families. The α, β, and γ tubulin family genes are found in all eukaryotes. α and β tubulin are the main components of microtubules, whereas γ tubulin plays an important role in the nucleation of microtubule assembly. There are numerous α and β tubulin genes, which are highly conserved among species.

(16) serine (or systemine) proteinase inhibitor, clade B, member 1a
It was first found in monocytes and neutrophils and was named for its strong inhibition of neutrophil elastase activity. It also inhibits pancreatic elastase and cathepsin G activities and has a wide range of protease inhibitory activities. It may be useful for the treatment of patients with cystic fibrosis.

(17) transdolase 1
This protein is the main enzyme supplying ribose 5-phosphate for nucleic acid synthesis and NADPH for lipid biosynthesis. This enzyme also acts to maintain glutathione in a depleted state, and as a result, it can protect cells by protecting sulfhydryl groups from oxygen radicals. This gene is thought to be involved in multiple sclerosis.

  The above protein may be a precursor protein or a mature protein, and may be a cleaved type or a non-cleaved type. Examples of the precursor protein include a proprotein and a preproprotein. Some proproteins and preproproteins have a signal peptide.

  In the method of the present invention, the expression of a specific protein in skin cells and / or skin tissue may be measured, or the gene expression thereof may be measured. For example, it can be measured by Northern blot method, RT-PCR method, Western blot method, immunohistochemical analysis method, ELISA method, antibody chip, cDNA microarray, fluorescence resonance energy transfer (FRET) measurement method, etc. it can.

In order to measure the expression of a specific protein at the protein level, an antibody that specifically recognizes the protein to be measured may be used. The antibody may be either a monoclonal antibody or a polyclonal antibody. These antibodies can be produced by known methods, and some are commercially available. When measured by Western blotting, the antibody is secondarily detected using ¹²⁵ I-labeled protein A, peroxidase-conjugated IgG, or the like. When measurement is performed by immunohistochemical analysis, the antibody may be labeled with a fluorescent dye, ferritin, enzyme, or the like.

  In order to measure the gene expression of a specific protein at the RNA level, a nucleic acid probe that can specifically hybridize with the mRNA of the protein to be measured may be used (when measured by Northern blotting). Alternatively, a pair of nucleic acid primers comprising a nucleic acid primer that can specifically hybridize with mRNA of a protein to be measured and a nucleic acid primer that can specifically hybridize with cDNA synthesized using the mRNA as a template may be used. Good (when measuring by PCR method). The nucleic acid probe and the nucleic acid primer can be designed based on the gene information of the protein to be measured. The nucleic acid probe is usually about 15 to 1500 bases. The nucleic acid probe may be labeled with a radioactive element, a fluorescent dye, an enzyme, or the like. The nucleic acid primer is usually about 15 to 30 bases.

  In the present invention, expression of a specific protein in skin cells and / or skin tissue and / or presence / absence of gene expression thereof may be detected, or the expression level may be measured. The presence or absence of expression of the protein and / or its mRNA can be confirmed by the presence or absence of the appearance of a spot or band at a predetermined position. The expression level of protein and / or mRNA thereof can be measured by the staining intensity of spots and bands. Alternatively, the protein and / or its mRNA may be quantified. In order to detect multiple gene expression and multiple protein expression at the same time, DNA array (probe is fixed to substrate) (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, DECEMBER 2002, 951-960), protein chip (antibody substrate) It is preferable to use a detection method such as (NATURE REVIEWS, DRUG DISCOVERY, VOLUME1, SEPTEMBER 2002, 683-695), Luminex (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, JUNE 2002, 447-456).

  The skin cells and skin tissue are preferably derived from the subject to be determined, and the biological species of the subject include humans, pigs, monkeys, chimpanzees, dogs, cows, rabbits, rats, mice and other mammals Animals can be mentioned.

  In order to determine the influence on wrinkle formation (suppression) by the method of the present invention, a skin biopsy sample, or cultured skin cells or cultured skin tissue obtained therefrom can be used. As the skin biopsy sample, a sample obtained by collecting the horny layer of the skin with a tape (horny layer checker) may be used.

  Examples of skin cells include epidermal keratinocytes, skin fibroblasts, Langerhans cells, melanocytes, mast cells, endothelial cells, sebocytes, hair papilla cells, hair matrix cells, and the like. Skin cells can be collected from the skin by a known method (new cell culture experimental method for molecular biology research, p57-71, Yodosha, 1999).

  Examples of the skin tissue include the stratum corneum, epidermis and dermis of the skin. The skin tissue can be collected from the skin by a known method (Acta. Derm. Veneleol., 85, 389-393, 2005).

  The skin biopsy sample may be a cell or a tissue. As the skin biopsy sample, a sample obtained by collecting the stratum corneum of the skin with a tape such as a stratum corneum checker may be used. The stratum corneum checker has been used in the past to collect stratum corneum samples for measuring the degree of keratinization and cell area of the stratum corneum and evaluating the degree of rough skin and the turnover speed of the stratum corneum. Advances and future prospects of usefulness / evaluation technology, Japan Cosmetics Engineers Association, Yakuji Nippo, p95-96). A stratum corneum sample can be easily and safely collected at a counseling store or at home, and it is very useful as a method for obtaining protein from the stratum corneum without being invasive.

The present invention also provides a method for identifying a substance effective for the treatment and / or prevention of wrinkle formation and suppression. This method comprises the following steps:
(a) contacting the test substance with skin cells and / or skin tissue;
(b) a step of culturing the skin cells and / or skin tissue brought into contact with the test substance in step (a) for a predetermined time;
(c) measuring the expression of a specific protein and / or its gene expression in the skin cells and / or skin tissue cultured in step (b), and
(d) including a step of evaluating the effect of the test substance by comparing the expression of the specific protein measured in step (c) with the expression of the specific protein before the action of the test substance.

  The test substance may be any substance, such as protein, peptide, vitamin, hormone, polysaccharide, oligosaccharide, monosaccharide, low molecular weight compound, nucleic acid (DNA, RNA, oligonucleotide, mononucleotide, etc.), lipid, other than the above Natural compounds, synthetic compounds, plant extracts, fractions of plant extracts, mixtures thereof, and the like.

The skin cells and skin tissues are as described above.
Contact between the test substance and skin cells and / or skin tissue may be by any method. For example, a method of adding a test substance to a culture solution of skin cells and / or skin tissue, a method of culturing skin cells and / or skin tissue on a culture container or a culture sheet to which the test substance is applied or fixed, etc. it can. Alternatively, a test substance may be directly applied to the skin or a test substance may be orally administered using a living body such as a human or a non-human mammal (eg, mouse, rat, guinea pig, rabbit, pig, etc.).

  The culture time of skin cells and / or skin tissue is not particularly limited. Any time may be used so long as the presence or absence of the effect of the test substance on the expression of a specific protein in the skin cells and / or skin tissue and / or its gene expression can be confirmed.

  The skin cell and / or skin tissue serving as a comparison control may be a skin cell and / or skin tissue before contact with the test substance. It may be a skin cell and / or skin tissue subjected to the same treatment except that the test substance is not contacted.

The specific protein is preferably selected from the group consisting of the following 17 types.
Group 1 (G1)
(1) peroxyredoxin 1,
(2) peptidylpropyl isomerase A,
(3) far upstream element (FUSE) binding protein 1
(12) ras-related nuclear protein
Group 2 (G2)
(4) Keratin 6A,
(5) Keratin 16,
(6) Keratin 17,
(7) Profilin 1,
(8) tryptophan-tRNA ligase,
(9) Protein disulphide-isomerase A6 precursor,
(10) fatty acid binding protein 5, epidermal,
(11) phosphoglycerate mutase 2,
(13) S100 calcium binding protein A9 (calgranulin B)
(14) electin, galactose binding, soluble 7
(15) tubulin, alpha 1
(16) serine (or systemine) proteinase inhibitor, clade B, member 1a
(17) transdolase 1

  As one example of the present invention, in the skin cells and / or skin tissue contacted with the test substance, the expression levels of the four types of group 1 (G1) are increased compared to the control, If it can be evaluated that there is an effect of increasing the expression of any protein, this test substance can be identified as a substance effective in suppressing or improving wrinkle generation.

  Further, as another example of the present invention, the expression level of 13 types of group 1 (G2) is decreased in the skin cells and / or skin tissue contacted with the test substance as compared with the control, and the test substance is If it can be evaluated that the expression of any of these proteins is reduced, this test substance can be identified as a substance effective in suppressing or improving wrinkle generation.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.

[Test 1-Wrinkle formation and topical administration test-]
In order to search for biomarkers that correlate with the phenomenon of “wrinkle formation”, a sample capable of qualitative evaluation of wrinkles is required. Since in vitro assay system using cultured cells cannot qualitatively evaluate wrinkles, induction of wrinkle formation by long-term UV irradiation using hairless mice, and suppression of wrinkle formation by compounds or collection of improved skin samples Went.

<Experimental animals, equipment, compounds>
(1) Experimental animal Hos; HR1 週 5 weeks old (Hoshino experimental material)
(2) Ethanol 99.8%
(3) Nenbutanol (Dainippon Sumitomo Pharma)
(4) Retinol VitaminA alcohol (ALEXIS)
(5) Ultraviolet irradiation device Ultraviolet A wave (FL32SBL / DMR: manufactured by Clinical Supply Co., Ltd.)
Ultraviolet B wave (FL32SE / DMR: manufactured by Clinical Supply Co., Ltd.)
(6) Replica collection tool
Reflective replica collection kit made by Asahi Biomed

<Wrinkle formation and topical administration test>
1. After acclimating Ares Mouse Hos; HR1 to Experimental Method (1) for 1 week, the mouse was exposed to low-intensity UV three times a week using a UV irradiation apparatus. The cumulative amount of UV irradiation at one time is 80 kJ / mm2 for UVA waves and 0.1 kJ for UVB waves. This UV irradiation work is carried out for 8 weeks (24 times in total), and drugs containing SB (referred to as “Silybin”), retinol, ethanol within 30 minutes after UV irradiation from the 8th week when fine wrinkles were observed. Each was treated on the back skin of the mice for 4 weeks. As a solvent (vehicle) in which various samples were dissolved, ethanol in which SB was dissolved was used.
After 12 weeks of irradiation (after 4 weeks of application), replicas were collected to accurately capture the appearance of mice and the formation of wrinkles. Thereafter, the skin was peeled off and stored frozen at −80 ° C., or a frozen section sample and a formalin fixed sample were prepared. An outline of the schedule is shown in FIG.

(2) Breeding environment
The animals were bred in a conventional breeding environment where 25 ° C ± 2 ° C, humidity 50% ± 5%, food was MR (Oriental Yeast), and tap water was freely available. Each group was divided into 5 groups and reared in the same cage. The sunshine set day and night every 12 hours from 7am to 7pm.

2. Group setting
The experiment was divided into 6 groups. All groups other than the group (group A) where only vehicle ethanol was applied without irradiation with ultraviolet rays were irradiated with ultraviolet rays. As a control group for wrinkle formation, a group (Group B) for UV irradiation and ethanol application was set. In addition, as the SB application group, three concentrations were set: 0.1% (Group C), 0.3% (Group D), and 1% (Group E). In addition, a group (F group) using 0.1% retinol was set as a positive control. One cage contained 5 mice from the same test group. The administration of the six groups and the external preparation is shown in Table 1.

3. When ultraviolet radiation UV irradiation, animals were transferred to special cages, it was irradiated with UVB20mJ / cm ² and UVA10J / cm ² by one group. Irradiation was carried out for 12 weeks in a three-day cycle of Monday, Wednesday and Friday.

4). Dissection After fasting for 18 hours from the day after the end of this breeding period in each group, anesthesia was introduced by intraperitoneal administration of Nembutal (40 mg / kg) for dissection. After confirming the effect of anesthesia, a photograph of the mouse skin was taken with a single-lens reflex camera (manufactured by NICON), and then a reflection-type replica was collected, followed by laparotomy and whole blood collection from the heart. Peel the entire back skin, remove muscle and subcutaneous tissue as much as possible, chop it into 1cm squares, and divide it into five 1.5ml tubes for proteome analysis and protein analysis using liquid nitrogen Immediately frozen. They were then placed in OCT compound for frozen tissue sections and frozen in isopentane cooled with liquid nitrogen. Next, it was immersed in mild form solution (WAKO) diluted twice with PBS for paraffin sections, shaken at 4 ° C. for one day and fixed, and then a paraffin block was prepared.

5). Tissue Staining Neutral formalin soaked skin tissue was embedded in paraffin according to a conventional method, and affixed to a glass slide with a thickness of 5 μm. Each skin section was subjected to hematoxylin-eosin staining (HE staining) according to a standard method.

6). Test progress
(1) Effect of silybin on photoaging-induced wrinkle formation model mice
I) UV irradiation 4 weeks, 8 weeks
At 4 weeks from the start of UV irradiation, the appearance of the UV-irradiated mouse skin was more reddish than that of unirradiated mice, and hair loss began to be observed. When irradiation was continued for 4 weeks (8 weeks in total), a fine wrinkle-like appearance was observed.
When the tissue image of the 8th week skin in which fine wrinkles were observed was examined using HE staining, thickening of the epidermis was observed. Epidermal thickening is one of the indicators used as an indicator of photoaging.
From the above, it was considered that photoaging was induced and fine wrinkles began to be formed, and treatment of the compounds of each group was started.

2) Test results of UV irradiation for 12 weeks, compound administration for 4 weeks (a) Appearance change, wrinkle formation inhibition The compound was administered externally and the appearance photograph after 4 weeks and the obtained replica image are shown in the next section. As shown in the appearance, deep and large wrinkles observed in mice treated with UV-irradiated ethanol were inhibited from being formed by applying SB or RN (retinol). In addition, RN treatment was considered to depend on skin irritation, but skin roughness and flaking were observed, whereas such skin changes were not observed in SB treatment.
Based on the obtained external appearance photo, Bissett, D. et al., Photodermatol. Photoimmunol. Photomed (1990) was used as a reference, and based on the grading index of wrinkles, it was digitized. The result is shown in FIG. The specific values of the wrinkle grades in FIG. 2 are 0.0 for group A (ethanol-irradiated UV non-irradiated), 7.0 for group B (ethanol-irradiated UV irradiated), 4.0 for group C (0.1% SB-coated UV irradiated), and D group. It was 2.8 in the 0.3% SB coating UV irradiation, 3.6 in the E group (1% SB coating UV irradiation), and 3.6 in the F group (0.1% RN coating UV irradiation). As a result, in the SB treatment group, 0.3% (group D) showed the most wrinkle suppressing effect, and no concentration dependency was observed. The reason for this is that in group E, SB solubility at 1% was very saturated, so precipitation occurred immediately when applied to the skin, and it did not penetrate deep into the skin, making it difficult to exert its effect. .

(B) Morphological change Next, after fixing using mild form, a section of a block embedded in paraffin was prepared, and HE staining was performed. When the obtained image was observed, thickening of the epidermis was observed in any of the samples irradiated with UV (UV irradiation EtOH treatment, UV irradiation SB treatment, UV irradiation RN treatment). The thickening of the epidermis observed in the UV-irradiated EtOH group tends to give a shielding effect on the epidermis by increasing the thickness of the epidermis in order to suppress the penetration of UV into the body as an indicator of photoaging. It is thought to be the result of a biological reaction. Moreover, it is considered that the thickening was most observed because retinol was caused by the improvement of cell proliferation ability by retinol as already reported elsewhere. In addition, with regard to SB treatment, no significant suppression of epidermal thickening, which was observed with other components with strong antioxidant power, was observed. This is a result consistent with the screening of retinol as a positive compound, and SB also has the ability of epidermal cells to proliferate in the same way as retinol, and it is considered that the effect was recognized in the form of thickening.
From the above results, we succeeded in obtaining skin with wrinkles formed by ultraviolet irradiation and a skin sample with wrinkle formation suppressed by silybin treatment of a wrinkle formation inhibitor. In the next step, proteome analysis, the skin of the skin sample group B (UV irradiation EtOH treatment) obtained in this experiment was wrinkled, and the D group (UV irradiation 0.3%) had the strongest wrinkle suppression effect. The experiment was carried out using SB treatment) without wrinkles.

[2. Test 2 Proteome analysis using photoaging-induced wrinkle formation and inhibition mouse skin]
<Experimental materials and methods>
1. Experimental material Mouse skin Out of the wrinkled group (group B) treated with only ethanol and the wrinkle-free group prepared by applying 0.3% SB, a remarkable effect can be confirmed from the photographed appearance photograph. Three animals each (3 animals with deeper wrinkles and 3 animals with more suppressed wrinkles) were selected and used as samples.

2. Experimental Method In this test, we performed a proteome analysis based on the 2D-DIGE method / mass analysis. The sample used for 2D DIGE method collected and used only the epidermis tissue, and prepared protein using LMD (laser microdissection) method or hot water bath method. The epidermis-specific proteome analysis is based on the originality of the present invention and is a completely new research method. After proteome analysis, validation by Western blotting was performed to verify variation.
In the 2D-DIGE method, comparison data (for example, whether or not silybin is applied) is obtained by developing the color of the protein under one condition in red and the protein under the other condition in green. When the protein expression level is the same, yellow (because red and green overlap), and when one of the protein expression levels is low or high, the color is red or green (because red and green are not balanced), so the expression level is This is a technique that can detect different proteins (not yellow, but red or green).
Next, the protein was identified using the mass spectrometer (LC-Q-TOF / MS) about the spot by which the fluctuation | variation was recognized. The obtained results were analyzed using a database (Mascot), and proteins considered to be candidates for fluctuating spots were determined.

<Photo-aging-induced wrinkle-forming protein specific proteome analysis test>
1. Dissection and collection of animal skin After fasting for 18 hours from the day after the end of the breeding period, anesthesia was introduced by intraperitoneal administration of Nembutal (40 mg / kg) to perform dissection. After confirming the effect of anesthesia, laparotomy and whole blood were collected from the heart. The entire back skin was peeled off, and the muscular tissue and subcutaneous tissue were removed as much as possible, and chopped into 1 cm squares. The hot water method proteome analysis and validation proteins were divided into five 1.5 ml tubes, immediately frozen using liquid nitrogen, and stored in a -80 ° C freezer. Next, in order to prepare frozen sections for LMD proteome analysis, they were sealed in about 1 ml of OCT compound (J-TEC), frozen in isopentane cooled with liquid nitrogen, and stored in a -80 ° C freezer.

2. LMD sampling method
The frozen skin sample embedded with the OTC compound, which had been stored at -80 ° C, was left in a cryostat at -10 ° C for 15 minutes. Thereafter, sections were prepared with a thickness of 10 μm using a cryostat. The obtained section was attached to a slide glass with a membrane film (Meiwaforsis). Next, the slide glass was fixed in 95% EtOH cooled at 4 ° C for 1 minute, washed with purified water for 1 minute, stained with Mayer's Hematoxilin stain (WAKO) for 1 minute, and washed again with purified water for 1 minute. The solution was replaced with 95% EtOH for 1 minute and 100% EtOH for another 1 minute, and the resulting slide glass was air-dried at room temperature for 1 hour. After air drying, a 2 mm ² epidermal portion was cut from about 4 slides using a Palm, Ltd. laser microdissection device, and made into a 500 ml tube. Next, the collection tube is centrifuged at 4 ° C, 13000 rpm for 10 minutes, and 50 ml of Lysis buffer {7 M Urea, 2M Thiourea, 4% CHAPS, 30 mM Tris-HCL (pH 8.0), 1% tritonx-100)}. Thereafter, Vortex treatment was performed for 30 seconds and ultrasonic treatment was performed 5 times for 15 seconds. Thereafter, the mixture was centrifuged at 13000 rpm for 20 minutes, the supernatant was taken out, and protein quantification was performed using a protein assay reagent (Bio-Rad).

3. Hot water bath method
Place the sample stored at -80 ° C in PBS (-) heated at 60 ° C, place in a water bath, place the ice-cooled glass plate face down, and use tweezers 10 seconds later. Only the epidermis part was collected. Place the collected epidermis in a 1.5 ml tube, add 300 μl of Lysis buffer {7 M Urea, 2M Thiourea, 4% CHAPS, 30 mM Tris-HCL (pH. 8.0), 1% tritonx-100)}, and vortex for 20 seconds. Then, handy pestle (TOYOBO) was connected as a pestle to a mini cordless grinder (Funakoshi), and the pestle was rotated at 9000 rpm to perform homogenization. Thereafter, sonication was further performed 5 times for 15 seconds, followed by centrifugation at 4 ° C., 13000 rpm for 20 minutes, the supernatant was taken out, and protein quantification was performed using a protein assay reagent (Bio-Rad).

4). Proteome analysis (2D-DIGE, mass analysis)
The obtained samples were each added with 20 nmol of TECP {Tris-(-2-carboxyethyl) phosphine hydoride} per 50 μg and reacted in the dark at 37 ° C. for 1 hour to cleave the SS bond of the cysteine residue of the protein. Thereafter, 40 nmol of Cye3 (saturation labeling, Amershamsha) per 50 μg of protein in the wrinkled group sample was added and reacted at 37 ° C. for 30 minutes to label the cysteine thiol group. Similarly, for wrinkle-free protein, 40 nmol of Cye5 (saturation labeling, Amershamsha) was added to 50 μg and reacted at 37 ° C. for 30 minutes to label the cysteine thiol group. After labeling, the same amount of 2 × sample buffer {7 M Urea, 2M Thiourea, 4% CHAPS, 30 mM, 2% DTT, 2% IPG buffer} was added and left on ice for 15 minutes to stop the reaction. After stopping the reaction, make the total volume to 500 μl using electrophoresis buffer {7 M Urea, 2M Thiourea, 4% CHAPS, 2 mg / ml DTT, 1% IPG buffer} for isoelectric focusing. Electrophoresis was performed. For electrophoresis, a dry strip of immobilon strip 24 cm / pH 3-10 (GE Healthcare) was used, and mineral oil was overlaid from above to prevent drying, and Ettan IPGphor (GE Healthcare) was used. Development conditions were such that the swelling was 12 hours, the voltage was 500 V for 1 hour, 1000 V for 1 hour, and 6000 V so that the total Vhr was 90000 Vhr. After the isoelectric focusing is completed, the immobilon strip is gently shaken for 15 minutes with paralleling buffer (6M urea, 50 mM Tris-HCl pH 8.0, 1% DTT, 2% SDS, 30% glycerol), and the gel concentration is reduced. This was applied to a polyacrylamide gel adjusted at 10% and subjected to second-dimensional electrophoresis. The gel after electrophoresis was read with Tyhoon 9400 (GE Healthcare). Cy3 label images were scanned with Ex540nm and Em590, and Cy5 label images were scanned with Ex620nm and Em680.
The comparative analysis was performed with Decyder software (GE Healthcare) using the obtained image. In the statistical analysis in the software, comparative analysis was performed in the wrinkle-free and wrinkle-free groups, and the spots that fluctuated significantly (P <0.05) were examined. For spots with significant fluctuations, perform in-gel enzyme digestion according to the method described in the literature (proteomics 2003, 3, 13181324) and analyze with LC-Q-TOF / MS. Identification was performed by analysis using the database MASCOT (http://www.matrixscience.com).

5). Validation (variation verification)
The collected mouse skin sample was frozen and pulverized using a cryopress (Microtech Nithion), powdered, and then skin lysis buffer {50 mM Tris-HCl (pH.7.5), 120 mM NaCl, 1 mM Na3VO4 , 0.4% NP-40, 1% Triton-X100} was added, and a mini cordless grinder (Funakoshi) was connected to Handy pestle (TOYOBO) as a pestle, and the pestle was rotated at 9000 rpm to perform homogenization. Thereafter, centrifugation was performed at 4 ° C., 13000 rpm for 20 minutes, the supernatant was taken out, and protein quantification was performed using a protein assay reagent (Bio-Rad). The obtained protein solution is adjusted to 2 mg / ml using skin lysis buffer, and an equal volume of 2 × SDS treatment solution {0.125 M Tris-HCl (pH 6.8), 10% glycerol, 3% SDS, 10% mercapto Ethanol} was added, and heat block treatment was performed at 95 ° C. for 5 minutes, and this was subjected to SDS-PAGE using a Laemmli Tris-Glycin system as a sample for Western blotting. After SDS-PAGE, the protein in the gel is transferred to a PVDF membrane (Millipore) at a constant current of 0.8 mA per cm ² and then a blocking solution (skim milk 0.1% polyoxyethylene (20) to a concentration of 5%). Solution in PBS containing sorbitan monolaurate) and blocked at 4 ° C. overnight. After washing with a washing solution {PBS containing 0.1% Tween-20}, it was immersed in a primary antibody solution (specific antibody solution of each protein prepared in an appropriate amount with the washing solution) and allowed to react at room temperature for 1 hour.
The types and dilution ratios of primary antibodies used are as follows: Goat Anti-TRPRS polyclonal antibody (Santa Cruz Biotechnology, 2000 times dilution), Mouse Anti-keratin6 polyclonal antibody (abcam, 500 times dilution), rabbit Anti- keratin16 polyclonal antibody (abcam, diluted 1000 times), rabbit Anti-keratin17 polyclonal antibody (abcam, diluted 1000 times), chicken Anti-PDIA6 polyclonal antibody (abcam, diluted 2000 times), rabbit Anti-Profilin1 polyclonal antibody (abcam, 1000 times) Dilution), rabbit Anti-Prx1 polyclonal antibody (Affinity Bio Reagent, diluted 1000 times), rabbit Anti-PPIA polyclonal antibody (Protein Tech, diluted 1000 times), rabbit Anti-FUBP1 polyclonal antibody (AVIVA SYSTEMS Biology, diluted 1000 times), goat Anti-PGM2 polyclonal antibody (Everest Biotech, diluted 1000 times), Mouse Anti-RAN monoclonal antibody (Gene Tex, diluted 1000 times), goat Anti-calgranulin B polyclonal antibody (Protein Tech, diluted 1000 times), rabbit Anti-FABP5 polyclonal antibody (Protein T ech, 1000 times dilution), Mouse Anti-Tubulin α monoclonal antibody (Thermo, 2000 times dilution), rabbit Anti-SERPINB1 polyclonal antibody (abcam, 1000 times dilution), goat Anti-translodase polyclonal antibody (Santa Cruz Biotechnology, 2000 times dilution) ), Rabit Anti-Galectin-7 polyclonal antibody (Gene Tex, diluted 2000 times). After washing, secondary antibody solution {Horseradish peroxidase labeled Anti-rabit IgG (GE Healthcare), Horseradish peroxidase labeled Anti-goat (GE Healthcare), Horseradish peroxidase labeled Anti-chicken IgM (Santa Cruz), diluted 5000 times with washing solution, Alternatively, it was immersed in 1000 times diluted True Blot Horseradish peroxidase labeled Anti-mouse IgG (eBioscience)} and allowed to react at room temperature for 1 hour. After washing, light was emitted using an ECL plus western blotting detection reagent (GE Healthcare) and exposed to an X-ray film. The blackening intensity exposed to the X-ray film was quantified using Image J (http://rsbweb.nih.gov/ij/), an image analysis software, and the relative expression level of each protein was calculated.

6). Experimental results (1) Electrophoretic results (a) Electrophoresis of skin samples obtained by the LMD method
About 2000 spots could be observed from the electropherogram of the skin sample obtained by the LMD method. The appearance of the bright spots appearing in the electropherogram is shown in FIG. In FIGS. 3A and 3B, the attached drawings are difficult to discriminate in monochrome display, but the actual observation is displayed in color and is discriminated as green and red. In this example, it was determined that green is “wrinkle” and red is “no wrinkle”.

(B) Electrophoresis of the skin sample obtained by the hot water method About 2500 spots were detected from the electropherogram of the skin sample obtained by the hot water method. Results are shown. The appearance of the bright spot in the electrophoretic diagram is shown in FIG. 3B for reference.

(2) Two-dimensional difference gel electrophoresis analysis results (2D DIGE)
The electropherogram obtained by the 2D DIGE method was analyzed with the software Decyder (GE Healthcare), and spots with changes were found in the wrinkle-free group.
(A) Results of analysis of skin samples obtained by the LMD method
As a result of analysis by Decyder (GE Healthcare), six spots whose expression was significantly changed 1.5 times or more or 1/2 or less were obtained. The breakdown was 6 spots where the expression level decreased due to wrinkle suppression by silybin. As shown in FIG.
(B) Analysis results of skin samples obtained by the hot water method
As a result of analysis by Decyder (GE Healthcare), 14 spots whose expression was significantly changed 1.5 times or more or 1/2 or less were obtained. The breakdown was 4 spots with increased expression due to suppression of wrinkles by silybin and 10 spots with decreased expression. As shown in FIG.

(3) Identification of fluctuating spots by mass spectrometer
Spots with fluctuations were extracted from the gel by Decyder analysis, digested in gel with trypsin, and then protein was identified using LC-Q-TOF / MS. After analyzing the obtained data peaks, protein identification was performed using Mascot search and database information. Krt6, Krt16, Krt17, Pfn1, Wars, Pdia6, Prdx1, Ppia, Fubp1, Fabp5, Pgm2, Ran, respectively. , S100A9, Lgals7, Tuba1, Serpinb1, Taldo1 (Table 2). Further, the positions of spots where fluctuations are recognized are shown in FIGS. 4 (a) and 4 (b). Changes in the expression level are shown in FIGS. Fig. 5 shows the proteins of 6 epidermis samples obtained by the LMD method, and Figs. 6 and 7 show 11 epidermis samples obtained by the water bath method (among 14 proteins, 3 proteins overlapping with the LMD method, keratin 6A, keratin 17, and keratin 16 are excluded).
The increased four types of proteins found from the results of the above (2) and the results of this identification were expressed as group 1 (G1), and the decreased 13 types of proteins were expressed as group 2 (G2).
The four proteins of Group 1 (G1) are the spots in Table 2.
No. 7 peroxyredoxin 1,
No. 8 peptidylpropyl isomerase A,
No. 9 far upstream elements (FUSE) binding protein 1,
No. 11 ras-related nuclear proteins
It is.
The 13 proteins of Group 2 (G2) are the spots in Table 2.
No. 1 Keratin 6A,
No. 2 protein disulphide-isomerase A6 precursor,
No. 3 Keratin 17,
No. 4 profile1,
No. 5 tryptophan-tRNA ligases,
No. 6 Keratin 16,
No. 10 phosphoglycerate mutase 2,
No. 12 S100 calcium binding protein A9 (calgranulin B),
No. 13 electins, galactose binding, soluble 7,
No. 14 fatity acid binding proteins 5, epidermal,
No. 15 tubulins, alpha 1,
No. 16 serine (or systemine) proteinase inhibitor, clade B, member 1a,
No. 17 transdolase 1
It is.

[2. Test 2 Proteome analysis using photoaging-induced wrinkle formation and inhibition mouse skin]
For proteins that were found to vary by proteome analysis, commercially available specific antibodies were purchased and verified by Western blotting (WB). The results of WB are shown in FIGS. 8 (a) and 8 (b). Next, the blackened image of the X-ray film obtained by WB was digitized using Image J and analyzed. The obtained WB results and analysis results are shown. The 11 proteins obtained by the water bath method shown in FIG. 8 (b) are the 14 proteins obtained by the water bath method except for 3 proteins overlapping with the LMD method, keratin 6A, keratin 17, and keratin 16 It is.
As a result of the experiment, all the proteins that were verified (Krt6, Krt16, Krt17, Pfn1, Wars, Pdia6, Prdx1, Ppia, Fabp1, FABP5, Pgm2, Ran, S100A9, Lgals7, 1 Tubad, 1 Reproducibility was observed. In other words, fluctuations in proteome analysis could be verified, and 17 types of wrinkle marker candidates that fluctuated with and without wrinkles were successfully obtained. In addition, if these proteins are detected using specific antibodies, it is considered possible to easily determine the wrinkle or photoaging state.

  A protein whose expression increases and decreases as wrinkles are formed or suppressed has been found. By examining the expression variation of these proteins, it becomes possible to make a more accurate diagnosis related to the causes and symptoms of wrinkles and to determine the risk of onset. In addition, these markers can be used for the development of aging and photoaging treatments such as wrinkles and the development of preventive drugs, cosmetics and health foods.

Claims (3)

A wrinkle marker composed of any of the following 17 proteins.
(G1):
(1) peroxyredoxin 1,
(2) peptidylpropyl isomerase A,
(3) far upstream element (FUSE) binding protein 1,
(12) ras-related nuclear protein
Protein (G2) selected from the group consisting of 4 types:
(4) Keratin 6A,
(5) Keratin 16,
(6) Keratin 17,
(7) Profilin 1,
(8) tryptophan-tRNA ligase,
(9) Protein disulphide-isomerase A6 precursor,
(10) fatty acid binding protein 5, epidermal,
(11) Phosphoglycerate Mutase 2
(13) S100 calcium binding protein A9 (calgranulin B)
(14) electin, galactose binding, soluble 7
(15) tubulin, alpha 1
(16) serine (or systemine) proteinase inhibitor, clade B, member 1a
(17) transdolase 1
A protein selected from the group consisting of 13 types   A method for examining the effect of a test substance on the formation of wrinkles on a skin by examining changes in the expression level of a protein serving as a wrinkle marker according to claim 1. 3. The method according to claim 2, wherein the wrinkle formation factor is ultraviolet exposure.