JP5027528B2 - Test method for skin aging - Google Patents

Description

  The present invention relates to a method for examining skin aging based on increase or decrease in gene expression. In particular, the present invention relates to a technique for determining photoaging of skin and skin modulation caused by ultraviolet irradiation or the like.

Skin changes in homeostasis due to various stress loads such as ultraviolet rays, sunlight, smoke exposure, metabolic abnormalities, dryness, aging, biological clock cycle abnormalities, house dust and environmental hormones, wrinkles and acne , Skin conditions such as rough skin, psoriasis and atopy, ie, phenotype worsen. A phenotype refers to a visible characteristic of an organism that arises from the interaction of a genotype with the environment.
A normal mammalian cell expresses about 20,000 to 30,000 out of more than 40,000 genes contained in its genome. Many genes are ubiquitously expressed. There are also non-universally expressed genes that differentiate cells and change cell functions.
In skin conditions such as wrinkles, itching, acne, rough skin, psoriasis and atopy, i.e. abnormal phenotypes, the pattern of gene expression in tissues and cells changes compared to the expression pattern seen in normal skin conditions There is a possibility.

Conventionally, these phenotypic changes have been evaluated by non-invasive techniques such as skin moisture content, lipid content and composition, and skin pH measurement, starting with appearance assessment. In addition, biopsy of skin is used to evaluate the expression of specific genes using RT-PCR, real time RT-PCR or Northern blottng, and multiple gene expression using SAGE (Serial Analysis of Gene Expression) It is evaluated. The protein expression level is limited to the type of antibody present, but in addition to collagen and MMPs, expression of laminin, integrins, interleukins, etc. that are thought to be involved in cell differentiation and inflammation is expressed by ELISA (Enzyme linked immuno solvent assay). ), Western blotting, or immunohistological staining techniques, and biomarkers for evaluating skin conditions have been found.
Regarding genetic changes associated with skin (light) aging, changes in steady-state mRNA levels of fibrillin have been demonstrated in solar elastic fibrosis (Bernstein et al., 1994). In addition, keratinocytes present in epidermal tissue that mediates contact with the dermis and basement membrane secrete many mediators that can stimulate fibroblast synthesis activity in response to UVA and / or UVB wave irradiation. However, some of them, such as TGF-β, IL-1β, and IL-10, have been shown to activate the promoter of the elastin gene and increase the accumulation of elastin (Kahari et al., 1992; Mauviel). Et al., 1993; Reitamo et al., 1994).
In recent years, studies using microarrays capable of comprehensively capturing gene expression for the same sample have been conducted. A microarray is a solid-phase system containing oligonucleotide sequences corresponding to thousands to tens of thousands of individual genes. Microarrays are used to test tissue or cell culture extracts, which genes are on or off in response to treatment, injury, age, sex, ethnicity, drugs, food, and cosmetics, and The degree of expression variation can be determined. Variations in gene expression occur prior to the expression of the protein that is the gene product and post-translational modification of the protein, and are extremely important in analyzing the initial response of the organism.

It has been found that by irradiating epidermal keratinocytes with ultraviolet B waves, heat shock protein (HSP) -27 gene expression is increased, while keratin 6 and keratin 17 gene expression is decreased (non-patented). Reference 1). However, the cDNA array, which is the test method of Non-Patent Document 1, must prepare a large number of probe clones for spotting, and can measure a fluctuating gene group depending on the selection of clones for expression analysis. Maybe not likely.
Moreover, as a result of irradiating the human buttock skin keratome with 4 MED ultraviolet rays and analyzing the gene expression, 18 gene markers whose expression increases and 17 gene markers whose expression decreases are found (Patent Document 1: JP 2005-520483 A). In addition, also in (Patent Document 2: Japanese Translation of PCT International Publication No. 2004-526410), a genetic marker that changes when ultraviolet rays are exposed to cells has been found. Monoclonal antibodies recognizing human aging marker protein (human SMP) are disclosed in Patent Document 3 (Japanese Patent Application Laid-Open No. 8-319298), a method for detecting a new human aging or human stress marker and a detection reagent, Patent Document 4 (Japanese Patent Laid-Open No. 2002-107363) discloses the use of an antibody against αA crystallin in which Asp ¹⁵¹ is a βD form.
However, these are analyzing changes in gene expression in the state of being photoaged, and it is unclear whether changes in phenotype can be predicted when the phenotype has not changed.

JP-T-2005-520483 JP-T-2004-526410 JP-A-8-319298 JP 2002-107363 A Bernd Becker, Thomas Vogt, Michael Landthaler, and Wilhelm Stolz, “Detection of Differentially Regulated Genes in Keratinocytes by cDNA Array Hybridization: Hsp27 and Other Novel Players in Response to Artficial Ultraviolet Radiation” THE JOURNAL OF INVESTGATIVE DERMATOLOGY VOL.116 NO.6 JUNE 2001, p. 983-988

  The subject of this invention is providing the method of test | inspecting the aging of the skin using the gene used as a skin aging marker. In particular, it relates to photoaging of the skin.

The main configuration of the present invention is as follows.
(1) A method for examining skin aging, which comprises measuring β- catenin.
(2) The method for examining skin aging according to (1), wherein the skin aging is photoaging.
(3) The method according to (1) or (2), wherein the measurement of β-catenin is by ELISA.

Examination of skin aging using 25 human genes shown in (A1) to (A10) and (B1) to (B15), which are marker genes for photoaging mainly due to skin aging, particularly ultraviolet exposure Could provide a way. By utilizing this marker gene, it is possible to determine skin aging, in particular, skin modulation due to photoaging caused by ultraviolet irradiation or the like.
In addition, by using this marker gene, it can be used for searching for a compound that suppresses skin modulation or aging caused by ultraviolet irradiation.
Using the mouse gene 29 corresponding to these 25 human genes and the mouse gene 29 in which four genes (A11) and (B16) to (B18) are further added, modulation of the skin by UV irradiation, aging, etc. It can utilize for the search of the compound to suppress.
In the present invention, each gene is specified as an index, but a protein produced corresponding to each gene can be used as an index in the same manner as a gene focused on. For example, it is exemplified that β-catenin, which is a product of gene (B1) GenBank Accession No. NM — 007614, and / or melanoma antigenic family A1, which is a product of gene (B4) GenBank Accession No. BB794642, can be used as effective indexes. .

In the research process of the present invention, the skin aging phenotypes “wrinkle”, “skin moisture”, “8-hydroxy-2′-deoxyguanodine production”, and “carbonyl oxide protein” are used as indices. We searched for skin aging gene markers by focusing on genes whose gene expression changes at the time when they do not change externally.
The genetic marker for determining the progress of skin aging needs to continue to increase or decrease as the skin aging progresses.
According to the present invention, a gene group (List1) (A1) to (A11) whose gene expression continues to increase during the process of skin aging and a gene group (List2) (B1) to (B18) whose expression continues to decrease are found. It was. “Accession No.” is an identification number of each gene in GenBank (NCBI nucleic acid sequence database), and Probe Set ID is an identification number unique to Gene Chip Expression Array manufactured by Affymetrix. Table 1 and Table 2 show the correspondence between human genes and mouse genes. Unless otherwise stated, the gene symbols shown below are represented by mouse genes.
In addition, the present invention can use each gene specified as an index and a protein produced corresponding to each gene as an index in the same manner as a gene focused on. For example, it is exemplified that β-catenin, which is a product of gene (B1) GenBank Accession No. NM — 007614, and / or melanoma antigenic family A1, which is a product of gene (B4) GenBank Accession No. BB794642, can be used as effective indexes. .

The gene group (List1) whose gene expression continues to increase during the skin aging process is the following 11 genes (A1) to (A11).
(A10) 6-phosphofructo-2-kinase, also known as fructose-2,6-biphosphatase 3, is a gene whose Accession No. is NM_133232, and has fructose-2,6-bisphosphate 2-phosphatase activity and ATP binding activity as an enzyme. is there.
(A5) dystrophin and muscular dystrophy are genes whose Accession No. is NM_007868 and encode an insoluble protein involved in muscle development.
(A8) CyclinH is a gene of Accession No. NM_023243 and is one of the components of the transcription factor complex called TFIIH that is involved in the nucleotide excise repair (NER) process, and is closely related to DNA damage. is doing.
(A9) SMC6 structural maintenance of chromosomes 6-like 1 is a gene of Accession No. AU022584, which is a gene having ATP binding activity localized in the cell nucleus.
(A6) RIKEN cDNA 1600014C10 gene is a gene whose Accession No. is BC019834 and has no known function other than interacting with the cell membrane.
(A1) COX15 homolog, cytochrome c oxidase assembly protein is a gene whose Accession No. is BC021498.
(A11) RIKEN cDNA 2310007F12 gene is a gene whose Accession No. is BG070464.
(A2) Hyperparathyroidism 2 homolog is a gene whose Accession No. is BB622571.
(A3) The microtubule-associated protein, RP / EB family, member 1 is a gene whose Accession No. is BB464192, and the protein that is the gene product has microtubule binding activity, such as cell movement, cell cycle, cell division, etc. Closely related to biological phenomena.
(A4) myosin IXa is a gene whose Accession No. is BB524436, and its gene product has a GTPase activation action and a diacylglycerol binding activity and forms a cytoskeleton.
(A7) F-Box protein 3 is the gene of Accession No. AV024918, and its gene product acts as an acceptor that recognizes the oxidized protein in the process in which the oxidized / senescent protein is ubiquitinated and disposed of in the lysosome. MyosinIXa (A4) and Dystrophin (A5) are proteins that are ubiquitous in muscle tissue, and it has recently been found that wrinkles are associated with changes in muscles deeper than skin tissue.

The genes (List2) whose expression continues to fall are the following 18 (B1) to (B18).
(B15) zinc finger and BTB domain containing 16 is the gene of Accession No. AA419994, and the protein of the gene product is a transcriptional control complex localized in the cell nucleus involved in negative feedback control of cell growth, control of apoptosis, etc. One of the constituent factors.
(B1) βcatenin is a gene of Accession No. NM — 007614, has binding activity with DNA and other proteins, particularly binding activity with cadherin, an adhesion molecule, and other cell-cell adhesion, transcription factor activity As a major molecule of TGFβ signaling pathway and Wnt / βcatenin signaling pathway, it is not limited to skin tissue but is involved in aging and canceration of each tissue.
(B13) transformation related protein 53 binding protein 1 is a gene whose Accession No. is AJ414734, and the protein that is a gene product has activities such as DNA repair and DNA-dependent transcriptional control.
(B14) WD repeat domain 22 is a gene whose Accession No. is BC027227, and the function of the protein that is its gene product has not been clarified.
(B2) RIKEN cDNA 1110037N09 gene is the gene whose Accession No. is BE197381, and the function of the protein that is the gene product has not been clarified.
(B3) DnaJ (Hsp40) homolog, subfamily A, member 3 is the gene of Accession No. AK007852, and the protein that is the gene product is a heat shock protein involved in protein folding.
(B4) The melanoma antigen is a gene whose Accession No. is BB794642, and the protein of the gene product corresponds to the change / motility of melanoma, and is closely related to pigmentation and canceration of skin tissue.
(B16) RIKEN cDNA 5730439E10 gene is a gene whose Accession No. is BQ031255, and the function of the protein that is the gene product has not been clarified.
(B5) AP2 associated kinase 1 is a gene of Accession No. BG067888, and the function of the protein that is its gene product has not been clarified.
(B18) RIKEN cDNA 2700060E02 gene is a gene whose Accession No. is AV167877, and the function other than that the gene product protein is expressed in the cell nucleus has not been clarified.
(B6) G-rich RNA sequence binding factor 1 is a gene whose Accession No. is BB223437, and the protein that is the gene product has an activity to bind to nucleic acids such as RNA, and acts as a signal peptide of transcription factor (TFIIB). To do.
(B7) The gap junction membrane channel protein alpha 1 is the gene whose Accession No. is AV330726, and the protein that is the gene product is called Connexin 43 (Gap junction 43 kDa heart protein). Involved in signal transduction between.
(B8) mitogen activated protein kinase kinase kinase 12 is an enzyme whose Accession No. is BB370469, and the protein that is the gene product is an enzyme that catalyzes the phosphorylation of amino acid residues such as serine, threonine, and tyrosine of the protein, It also has magnesium ion binding activity and ATP binding activity.
(B17) GPI-anchored membrane protein 1 is a gene whose Accession No. is BE981338, and the function of the protein that is the gene product is unclear.
(B9) Glucose phosphate isomerase 1 is a gene whose Accession No. is BB271021, and the protein that is the gene product is an enzyme that catalyzes the isomerization reaction of glucose-6-phosphate to fructose-6-phosphate. Also shows activity and growth factor activity.
(B10) Myocyte enhancer factor 2A is a gene whose Accession No. is AV255689, and the protein that is the gene product has DNA-binding activity and transcription-dependent activity in a DNA-dependent manner.
(B11) cDNA sequence AB023957 is a gene whose Accession No. is BB271021, and the function of the protein that is the gene product is unclear.
(B12) Mitochondrial ribosomal protein L52 is a gene whose Accession No. is AV024918, and the protein that is the gene product is one of the ribosome components.

In particular, these 29 genes are skin aging gene markers associated with photoaging of the skin, which is mainly caused by ultraviolet exposure.
The expression level of these 29 genes is compared with the skin of the part not directly exposed to sunlight, such as the stomach, or the skin of the part rarely exposed to the skin of the hand or face that is routinely exposed to sunlight. Thus, it is possible to determine the modulation of the skin exposed to ultraviolet light.

Expression of at least one of the marker gene sets selected from these 29 genes is either (i) a non-UV exposed control or (ii) UV exposed in skin exposed to UV radiation. It can be determined whether is modulated relative to a control that is shielded or attenuated. When applied to an individual, the individual is identified as sensitive or very sensitive to photo-damage or photoaging of the skin and aging by determining the modulation in at least one gene expression Is possible.
Sensitivity to changes in the external environment such as light and ultraviolet rays varies depending on the individual (race, gender, lifestyle). The gene group disclosed by the present invention lists genes whose expression continues to increase / decrease due to the stimulation of ultraviolet rays. By measuring the expression state of these genes in an individual, "how much is the skin aging state?" It is possible to provide a device such as a “skin aging marker gene expression measurement kit” that can predict whether “it is progressing” or “how much skin aging will progress by accumulating future life”. .
Gene expression analysis methods include cDNA microarray, SAGE (Serial Analysis of Gene Expression), RT-PCR, Northern Blotting and the like. Among them, cDNA microarray is convenient and effective as a measurement kit.
As a skin aging marker gene expression measurement kit, 29 individual genes can be measured one by one by a technique such as PCR. It is also possible to adopt a form of Gene Chip (Affymetrix).
However, a skin aging marker gene expression measurement kit that measures 29 individual genes one by one using a technique such as PCR is not efficient because it repeats the same operation, and the Gene Chip (Affymetrix described in this specification). In the application of the technique (2), it is troublesome to search for 29 genes one by one from the data of more than 22,000 genes, and the cost for one measurement becomes very expensive.

As another skin aging marker gene expression measurement kit, the progress of skin aging and photodamage can be simplified by preparing a spot-type cDNA microarray that can measure all 29 genes or a part of gene expression analysis. It is possible to evaluate correctly and quickly.
The skin aging marker gene expression measurement kit can measure not only skin but also skin substitutes. As a skin substitute, co-culture with skin constituent cells damaged by the overlaid collagen gel, improve the commercially available skin three-dimensional skin model and use it as a simulated artificial skin in the screening system, or a biological component There is known a method for differentiating the production-inhibiting action of advanced end glycation produced by incubating reducing sugar and protein in a solution system. In particular, in an in vitro screening for an active ingredient of a skin external preparation, a method for evaluating the safety and effectiveness of the skin external preparation constituents can be used using a three-dimensional skin model. For example, LSE (Living Skin Equivalent), which is a fully simulated skin model of human skin, is composed of an upper layer composed of human epithelial cells differentiated and multilayered, and a lower layer in which human skin fibroblasts are embedded in a collagen gel. Yes. In addition, the skin of mammals such as rats, mice, guinea pigs and pigs (especially Yucatan minipigs) in non-human species can also be used as an alternative, and various environmental factors (UV, sunlight, smoke exposure, metabolism, etc.) It is effective when evaluating the influence of the skin on abnormalities, dryness, aging, abnormal biological clock cycles, stress such as house dust and environmental hormones).
In the present invention, gene expression can be evaluated by measuring a protein that is a gene product. For example, it can be measured using Western blotting, immunohistochemical analysis, ELISA, antibody array and the like.
In order to measure the amount of protein produced, 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.

  These genes can be used for searching and screening for candidate substances exhibiting anti-aging effects. These 29 genes are effective as a screening tool for anti-aging compounds by ingesting tocopherol (vitamin E) and CoQ10, which are known as anti-aging agents and exhibit anti-oxidation effects, and compare with non-ingestion. I was able to confirm the sex.

Example 1
When hairless mice are irradiated with ultraviolet rays of the daily life level, wrinkles are formed after about 10 weeks. The gene expression over time in the wrinkle formation process was measured. Focusing on genes that change before the phenotype worsens, we extracted genes that serve as markers.

I. Experimental animals I-1. Species / strain / sex Species: mouse, strain: Hos: HR1 (hairless mouse), sex: female I-2. Breeding start age
5 weeks of age (starting UV irradiation at 6 weeks of age after 1 week of breeding)
I-3. Microorganism grade
SPF
I-4. Breeder Shimizu Experimental Materials Co., Ltd.

II. Breeding environment setting temperature and humidity: 24 ± 1 ℃, relative humidity 55 ± 5%
Air-conditioning equipment: All-fresh system Lighting time: 12 hours Automatic on / off system Breeding equipment: 5 plastic cages / cage Feeding: Powdered sterilized feed MF-1 (Oriental Yeast Industry Co., Ltd.), MF-1 A feed containing 0.05% by weight of tocopherol or CoQ10 was also prepared separately.
Administration of test subject: Ad libitized as a powdered diet. Fasted 18 hours before dissection.
Water supply: Free intake of sterilized tap water

III. Equipment used for UV irradiation
Portable irradiation equipment for UV-B and UV-A waves (manufactured by Central Medical)
UV-A wave irradiation lamp (FL20S / E-30 / DMR Clinical Supply)
UV-B wave irradiation lamp (FL20S / BL / DMR Clinical Supply)

IV. Test condition IV-1 UV irradiation The animal was transferred to a dedicated container for each cage, and UV irradiation was performed in an unrestrained state.
UV irradiation per time
For UV-B waves, the cumulative amount is 20 mJ / cm ² (irradiation for 1 minute under the condition of UV intensity average 3.33 W / m ² ),
For UV-A waves, replace the UV lamps for UV-A waves and UV-B waves, respectively, with an integrated amount of 100 J / cm ² (irradiation for about 40 minutes under the condition of an ultraviolet intensity average of 45 to 50 W / m ² ). Carried out.
The accumulated amount of ultraviolet rays was measured using UV MONITOR MS-211-I (manufactured by Eihiro Seiki). Ultraviolet irradiation was performed every other day, and ultraviolet irradiation immediately before dissection was terminated 24 hours before dissection. In the case of UV irradiation group for 10 weeks, UV-B wave 700 mJ / cm ² and UV-A wave 3.5 kJ / cm ² are exposed.

IV-2 Interim observation and measurement At the start of UV irradiation (0wk, 6 weeks old), visual evaluation of wrinkle formation on the back every 2 weeks of UV irradiation, measurement of skin moisture content, measurement of 8-hydroxy-2'-deoxyguanosine Measurement of carbonyl oxide protein was carried out. The results of the UV irradiation group were compared with those of the UV non-irradiated group, and the significant difference test was performed by student's t-test (p <0.05).

IV-3 Dissection After measuring the body weight and skin moisture of an individual fasted from 18 hours before dissection, anesthesia was introduced by intraperitoneal administration of Nembutal anesthesia (40 mg / kg). Thereafter, photography and skin replicas were collected.
An incision was made in the abdomen, and heparin blood was collected from the heart. Next, after the macroscopic observation of the organ, the skin of the entire back of the buttock was removed from the liver and the back of the head. Regarding the back skin, 2 cm from the caudal root on the dorsal side of the skin tissue to the neck and 1 cm ^{2 of} 0.5 cm site of the skin from the lumbar vertebra were cut out, fixed with Buan, and embedded in paraffin to preserve the skin tissue. The other back skin was quickly put in aluminum foil, rapidly frozen in liquid nitrogen, and stored at -80 ° C for a long time.

V Specimen UV-irradiated 0 weeks (6 weeks old) 5 each UV-irradiated 2 weeks (8 weeks old) 5 each UV-irradiated 4 weeks (10 weeks old) 5 each UV-irradiated 10 weeks (16 weeks old) ) 5 animals each UV irradiation 10 weeks (16 weeks old) DL-α-tocopherol mixed 5 animals each UV irradiation 10 weeks (16 weeks old) CoQ10 5 animals each UV irradiation 2 weeks (8 weeks old) 5 animals each UV irradiation 4 weeks (10 weeks old) 5 each UV irradiation 10 weeks (16 weeks old) 5 each UV irradiation 10 weeks (16 weeks old) DL-α-tocopherol mixed diet 5 each UV irradiation 10 weeks (16 weeks old) ) CoQ10 mixed feed 5 each

VI Appearance change 0 weeks and 10 weeks without UV irradiation, 10 weeks with UV irradiation, 0.05 wt% DL-α-tocopherol mixed with UV irradiation for 10 weeks, 0.05 wt% CoQ10 mixed with UV irradiation for 10 weeks Mouse photographs of the group, 2 weeks, 4 weeks, 6 weeks and 8 weeks of UV irradiation were taken, and the progress of skin aging was evaluated by scoring wrinkles.
Score 0 No wrinkles score 1 Minor wrinkles score 2 Wrinkles score 3 Deep wrinkles

For samples with unclear judgment, an intermediate value of 0.5 was scored. Mean ± SD was determined for the scores of each group. The significant difference test between the two groups was performed by student's t-test (significance level p <0.05). The results are shown in FIGS.
As shown in FIG. 1, the formation of wrinkles progressed significantly in the group irradiated with ultraviolet light for 10 weeks compared to the group not irradiated with ultraviolet light. And in the group to which DL-α-tocopherol and CoQ10 were mixed, wrinkle formation was significantly suppressed as compared with the UV (+) group.
As shown in Fig. 2, wrinkle formation progressed with the transition of the ultraviolet irradiation period, but wrinkle formation was observed significantly after 4 weeks of UV irradiation, and on the appearance after 2 weeks of UV irradiation. No skin aging phenomenon was observed.
It is thought that the irradiation of ultraviolet rays causes oxidation and drying of the mouse skin, and wrinkles are formed with the involvement of muscle movement, but the mechanism is unclear. However, it has been found that the wrinkle state changes due to the difference in the ultraviolet irradiation period.

0 weeks ⇒ No UV irradiation (wrinkle not formed)
2 weeks ⇒ UV irradiation (wrinkle not formed)
4 weeks ⇒ UV irradiation (formation of wrinkles slightly)
10 weeks ⇒ UV irradiation (wrinkle formation)

VII Measurement of skin moisture content The moisture content was measured 3 times using a moisture checker (manufactured by SCARA), measuring 3 cm from the tail of the back to the neck and 0.5 cm from the lumbar spine to the right. . The measurement days were the test start date and the intermediate observation day, which were 2 weeks, 4 weeks, 6 weeks, 8 weeks after the ultraviolet irradiation and the day of dissection (10 weeks of ultraviolet irradiation).
The mean value ± SD was determined for the skin moisture content (%) of each group. The significant difference test between the two groups was performed by student's t-test (significance level p <0.05).
From FIG. 3, a significant decrease in the amount of moisture in the skin was observed after 4 weeks of UV irradiation, and the drying of the skin proceeded with wrinkle formation.

VIII Measurement of 8-hydroxy 2'-deoxyguanosine in skin tissue 8-Hydroxy 2'-deoxyguanosine was measured using an immunohistochemical technique. At the time of animal dissection, 2 cm from the caudal root of the back of the skin tissue toward the neck and 1 cm ² of the skin at a 0.5 cm site on the right side from the lumbar vertebra were cut out, fixed with Buan, and embedded in paraffin to preserve the skin tissue. A 3 μm-thick section was appropriately prepared, and deparaffinization and hydrophilization were performed based on known methods. Antigen activation was performed by microwave treatment for 5 minutes in 0.01 M citrate buffer (pH 6.0). After cooling to room temperature, the reaction was carried out with methanol containing 0.3% hydrogen peroxide at room temperature for 20 minutes to inhibit endogenous peroxidase. After washing with water and 10 mM PBS (-), the goat serum was subjected to microwave treatment for 5 minutes with 75-fold diluted 10 mM PBS (-), blocked, the serum was removed, and the primary antibody (N45.1: Nikken Zeil Co., Ltd.) The antibody was reacted by microwave treatment at 5 μg / ml for 20 minutes. After washing twice with 10 mM PBS (−), a biotinylated secondary antibody (biotinylated goat immunoglobulin M; manufactured by DAKO) diluted 300-fold was reacted with the antibody by microwave treatment for 5 minutes. The plate was washed twice with 10 mM PBS (−) and reacted with an ABC reagent (ABC-HRP; manufactured by Vectastein) by microwave treatment for 5 minutes. The reaction was performed at room temperature for 3 minutes and 30 seconds using DAB (3,3-diaminobenzidine tetrahydrochloride: manufactured by DAKO) as a coloring reagent. After washing with water, dehydration and encapsulation were performed based on a known method. The staining of the skin tissue was observed under a microscope. The obtained image is shown in FIG. After 4 weeks of UV irradiation, the nucleus of the epidermal cells is mainly colored. An image was taken in using Adobe Photoshop, and stained portions in a fixed area in the image were digitized by NIH Imaging.

The mean ± SD was determined for the numerical values in each group. The significant difference test between the two groups was carried out by the student's t-test (significance level p <0.05) after correcting the average value of each group with the UV (−) group as the reference value of 1.
As shown in FIG. 5, 8-hydroxy-2′-deoxyguanosine does not increase after 2 weeks of UV irradiation, but significantly increases after 4 weeks of UV irradiation. -Deoxyguanosine increased.

IX Measurement of oxidized protein in skin tissue Oxidized protein, ie carbonylated protein, is labeled after carbonylated protein using 2,4-dinitrophenylhydrazine (DNPH) that specifically binds to the carbonyl group of the protein caused by oxidative damage. , Using an anti-DNPH antibody that specifically binds to DNPH. A specific method is as follows. Cut 1 cm ² of the skin at the UV irradiation site, add 1 ml of 0.1 M Tris buffer (pH 7.5) containing PMSF at 4 ° C., homogenize, centrifuge at 12,000 g × 20 minutes, and filter the supernatant. And analyzed. The protein was converted to dinitrophenylhydrazine (DNPH) by a known method (Nakamura et al., Journal of Biochemistry, 119, 768-774, 1996). The DNPH protein was separated by SDS-PAGE, and transferred to a polyvinylidene difluoride (PVDF) membrane using a protein transfer device. After the transfer, the membrane was blocked at room temperature for 30 minutes in 5% skim at 4 ° C in a PBS (-) solution containing milk, washed with PBS (-), and reacted with anti-DNPH antibody at 4 ° C overnight. After washing, it was reacted with biotinylated anti-rabbit immunoglobulin G for 1 hour. After washing, the PVDF film was exposed using a fluorescence detection kit (ECL PLUS), and the image was transferred with a medical automatic developing device. Image analysis was performed using a transilluminator.
The mean ± SD was determined for the numerical values in each group. A significant difference test between two groups was performed by student's t-test (significance level p <0.05).

  As shown in FIG. 6, the carbonyl oxide protein did not increase after 2 weeks of UV irradiation, but increased significantly after 4 weeks of UV irradiation, and thereafter the carbonyl oxide protein increased as the UV irradiation period increased.

X Test for Gene Expression Analysis Two back skins out of 5 in each group were selected, and total RNA samples for gene expression analysis were extracted. For the selection, two of the individuals with the smallest distance among the five animals in each group were selected according to the value of skin moisture. That is, an animal individual having a median (intermediate value) of skin water content of each individual and an animal individual closest to the median were selected and used as gene expression analysis samples. The average of the two measurements was used for the analysis.
Total RNA was extracted from the back skin of the selected individuals. The dorsal skin was chopped and ground in the presence of liquid nitrogen, and dispensed into Eppendorf tubes every tens of mg of wet weight. Total RNA extraction was performed using an RNase easy mini kit (manufactured by QIAGEN) according to the usual protocol.

  After the treatment according to the protocol recommended by Affymetrix, the expression pattern in the skin tissue was examined by the DNA microarray method. Specifically, cDNA was synthesized from the extracted total RNA using “SUPERSCRIPT choice system for cDNA synthesis” (trade name, manufactured by Invitrogen), and “Bio Array High Yield RNA Transcribing Lipid Transfer Protocol” using this cDNA as a template. (Trade name, manufactured by Enzo Diagnostics) was used to synthesize biotin-labeled cRNA in a test tube. After this cRNA was fragmented, hybridization was performed with a DNA microarray (trade name “GeneChipMouse Expression Array 430A” (manufactured by Affymetrix)) and “hybridization oven” (trade name, manufactured by Affymetrix), and R- After the reaction with phycoerythrin-streptavidin and the washing operation were performed with “Fluidics station” (trade name, manufactured by Affymetrix), the fluorescence intensity was measured with “Gene Array Scanner” (trade name, manufactured by Affymetrix). The expression level of related genes was analyzed. The DNA microarray contains 34,323 genes and 39,015 transcripts among mouse genes whose base sequences are known (including those whose functions are not yet known). All the mouse genes whose functions are already known are on this chip. The detection sensitivity is 1: 100,000, which is measured by adding a labeled transcript derived from a mouse cDNA clone to a mouse total RNA sample and detecting it. In addition, 22,690 genes were detected in mouse skin-derived total RNA used in this test, and used for analysis of gene expression increase / decrease.

The expression level ratio of the test group to the control group was calculated for the detected gene.
The combinations of the test group and the control group are as follows.
Expression level ratio Test group: UV irradiation 2 weeks (8 weeks old)
Control group: 0 weeks without UV irradiation (6 weeks old)
Expression level ratio2. Test group: UV irradiation 4 weeks (10 weeks old)
Control group: UV irradiation 2 weeks (8 weeks old)
Expression level ratio 3. Test group: UV irradiation 10 weeks (16 weeks old)
Control group: UV irradiation 4 weeks (10 weeks old)
Expression level ratio4. Test group: Ultraviolet irradiation 10 weeks (16 weeks old) DL-α-tocopherol diet
Control group: UV irradiation 10 weeks (16 weeks old)
Expression level ratio 5. Test group: Ultraviolet irradiation 10 weeks (16 weeks old) CoQ10 diet
Control group: UV irradiation 10 weeks (16 weeks old)

The gene expression level is proportional to the fluorescence intensity captured by the scanner.
Those with numerical values of fluorescence intensity exceeding 153.3 were classified as P (Present), and those with 153.3 or less were classified as A (Absent). When calculating the expression ratios 1 to 3, if both of the two mice in the test group and the two mice in the control group are A (Absent), even if the ratio of the test group to the control group is calculated, Since it was not possible to determine significantly whether gene expression was increased or decreased, such genes were deleted from the analysis. When any of the two mice in the test group and the two mice in the control group was P (Present), it was determined that the change in gene expression could be evaluated and used for analysis. Based on this criterion, 16880 genes out of the 22,690 genes were deleted from the analysis, and 5810 genes were used in the analysis.

When the ratio of gene expression of the test group to the control group (expression ratio) is 1.5 times or more, the expression level is increased, when the expression level is 0.67 times or less, the expression level is decreased, and the expression level is more than 0.67 times and 1.5 Less than double was defined as no change in expression level. Since Tables 1 and 2 gene expression ratio in the logarithmically it denoted the bottom 2, increased expression by _Log 2 1.5 ≒ + 0.585 or _more, Log in 2 0.67 ≒ -0.578 or less Decreased expression.

  The genetic marker for determining the progress of skin aging needs to continue to increase or decrease as the skin aging progresses. Therefore, continuous expression of the gene in 0 weeks without UV irradiation (6 weeks old) → 2 weeks with UV irradiation (8 weeks) → 4 weeks with UV irradiation (10 weeks) → 10 weeks with UV irradiation (16 weeks) 11 genes (Table 1) in which the gene expression level increased in all of the expression level ratios 1 to 3 were extracted and used as skin aging markers. Furthermore, continuous expression of the gene in 0 weeks (6 weeks old) without UV irradiation → 2 weeks with UV irradiation (8 weeks old) → 4 weeks with UV irradiation (10 weeks old) → 10 weeks with UV irradiation (16 weeks old) , Ie, 18 genes (Table 2) in which the expression level of the gene decreased in all of the above expression ratios 1 to 3 were extracted and used as skin aging markers.

  By the way, among the 1698 genes whose expression level ratio 1 [test group: UV irradiation 2 weeks (8 weeks old), control group: UV non-irradiation 0 weeks (6 weeks old)] increased, continuous UV irradiation 10 weeks 11 genes with increased gene expression and 1821 genes with reduced expression ratio 1 were extracted from 18 genes with continuously decreased gene expression up to 10 weeks of UV irradiation. . Moreover, 2291 genes were evaluated that the gene expression level was not changed based on the result of the expression level ratio 1. An image of the expression level ratio pathway is shown in FIG.

  In Tables 1 and 2, “Accession No.” is an identification number of each gene in GenBank (NCBI nucleic acid sequence database), and Probe Set ID is an identification number unique to GeneChip Expression Array manufactured by Affymetrix. In (A1) to (A10) and (B1) to (B15), since the correspondence between the mouse gene and the human gene is known, the same reference numerals are used. (A11) and (B16) to (B18) are mouse genes since the corresponding human genes are not known. This correspondence can be known from, for example, NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/).

  In order to confirm whether the aging (wrinkle) inhibitory effect can be detected by a skin aging marker, the gene expression level of mice fed with high concentrations of DL-α-tocopherol and CoQ10, which are effective in preventing and improving wrinkles, was examined. . As shown in FIG. 1, when ultraviolet rays are irradiated for 10 weeks, the skin is in a state of “recognizing moderate wrinkles” to “recognizing deep wrinkles”. On the other hand, the skin of a mouse irradiated with ultraviolet rays for 10 weeks while feeding a 0.05 wt% DL-α-tocopherol mixed feed is in a state where “the occurrence of slight wrinkles is clearly recognized” and 0.05 wt. The skin of mice irradiated with UV light for 10 weeks while ingesting 1% CoQ10 mixed feed is in a state where "minor wrinkles are clearly observed" to "moderate wrinkles are observed" and skin aging ( Wrinkles) are suppressed. Therefore, the expression level ratio 4 [Test group: UV irradiation 10 weeks (16 weeks old) DL-α-tocopherol diet, control group: UV irradiation 10 weeks (16 weeks old)], expression level ratio 5 [Test group: UV irradiation 10 weeks (16 weeks old) CoQ10 diet, control group: ultraviolet irradiation 10 weeks (16 weeks old)] were measured.

  As a result, 4 genes out of 11 genes whose expression levels increase with skin aging in Table 1, (A1) Accession No. BC021498 gene name COX15 homolog, cytochrome c oxidase assembly protein (yeast), (A2 ) Accession No. BB622571 gene name hyperparathyroidism 2 homolog (human), (A3) Accession No. BB464192 gene name microtubule-associated protein, RP / EB family, member 1, (A4) Accession No. BB524436 gene name myosin IXa expression level The ratios 4 and 5 are both −0.578 or less, and the expression level of the skin aging marker is suppressed in response to the suppression of aging (wrinkle) by DL-α-tocopherol intake and CoQ10 intake. It was.

  3 out of the 11 genes (A5) Accession No. NM — 007868 Gene name dystrophin, muscular dystrophy, (A6) Accession BC019834 Gene name RIKEN cDNA 1600014C10 gene, (A7) Accession No. AV024918 Gene name F-box only protein 3 The expression level ratio 4 was −0.578 or less, and the expression level of the gene decreased and the expression of the skin aging marker was suppressed according to the suppression of aging (wrinkle) by ingesting DL-α-tocopherol. However, the expression level ratio 5 was more than −0.578 and less than +0.585, and the gene expression level did not change.

  Among the 11 genes, the remaining 4 genes (A8) Accession No. NM_023243 Gene name cyclin H, (A9) Accession No. AU022584 Gene name SMC6 structural maintenance of chromosomes 6-like 1 (yeast), (A11) Accession No. BG070464 Gene name RIKEN cDNA 2310007F12 gene, (A10) Accession No. NM_133232 Gene name 6-phosphofructo-2-kinase / fructose-2, 6-biphosphatase 3 expression ratios 4 and 5 are both -0.578 It was less than +0.585, and the gene expression level did not change. None of the genes in Table 1 promoted the expression of a skin aging marker (an increase in gene expression level) with aging (wrinkle) suppression.

  In addition, 14 genes out of 18 genes whose expression level decreases with skin aging in Table 2 (B1) Accession No. NM — 007614 gene name catenin beta, (B2) Accession No. BE197381 gene name RIKEN cDNA 1110037N09 gene (B3) Accession No. AK007852 gene name DnaJ (Hsp40) homolog, subfamily A, member 3, (B4) Accession No. BB794642 gene name melanoma antigen, (B16) Accession No. BQ031255 gene name RIKEN cDNA 5730439E10 gene, (B5) Accession No. BG067888 Gene name AP2 associated kinase 1, (B6) Accession No. BB223437 Gene name G-rich RNA sequence binding factor 1, (B7) Accession No. AV330726 Gene name gap junction membrane channel protein alpha 1, (B8) Accession No. BB370469 Gene name mitogen activated protein kinase kinase kinase 12, (B17) Accession No. BE981338 Gene name GPI-anchored membrane protein 1, (B9) Accession No. BB271021 Gene name glucose phosphate isomerase 1, (B10) Accession No. AV255689 gene name myocyte enhancer factor 2A, (B11) Accession No. BB271021 gene name cDNA sequence AB023957, (B12) Accession No. AV024918 gene name mitochondria ribosomal protein L52 expression level ratio 4 and 5 was +0.585 or more, and the expression level of the gene was increased and the expression of the skin aging marker was suppressed according to aging (wrinkle) suppression by DL-α-tocopherol intake and CoQ10 intake.

  Of the 18 genes, 3 genes (B13) Accession No. AJ414734 gene name transformation related protein 53 binding protein 1, (B14) Accession BC027227 gene name WD repeat domain 22, (B15) Accession No. AA419994 gene name zinc finger and The expression level ratio 4 of BTB domain containing 16 is +0.585 or more, and the expression level of the gene increases and the expression of the skin aging marker is suppressed according to the aging (wrinkle) suppression by DL-α-tocopherol intake. It was. However, the expression level ratio 5 was more than −0.578 and less than +0.585, and the gene expression level did not change.

  Among the 11 genes, the remaining 1 gene (B18) Accession No. AV167877 gene name RIKEN cDNA 2700060E02 gene expression ratios 4 and 5 are both more than −0.578 and less than +0.585, The gene expression level did not change.

None of the genes in Table 2 promoted the expression of a skin aging marker (decrease in the gene expression level) with aging (wrinkle) suppression.
Therefore, 11 types of genes whose expression levels continue to increase with UV irradiation and 18 types of genes whose expression levels continue to decrease with UV irradiation are used as skin aging markers, thereby detecting the aging (wrinkle) suppression effect. I was able to confirm the possibility.
This result shows that in mice fed with high concentrations of DL-α-tocopherol and CoQ10, the 11 genes (A1) to (A11) suppressed the increase in the expression level compared to the non-intake comparative example. Moreover, since it was confirmed that the 18 types of genes (B1) to (B18) suppressed the decrease in the expression level, it should be effective as a screening means for substances having a wrinkle prevention / amelioration action. Is recognized. By using this screening means, it can be used to search for candidates for compounds, substances and extracts effective as an anti-aging action.

  In the transition of the skin appearance (wrinkle score) in Fig. 2, wrinkles are not formed at all in 2 weeks of UV irradiation, wrinkles are slightly formed in 4 weeks of UV irradiation, and wrinkles are completely formed in 10 weeks of UV irradiation. Is shown.

  In contrast, FIG. 3 shows changes in skin water content, FIG. 5 shows changes in skin tissue 8-hydroxy-2′-deoxyguanosine (8-OH dG), and FIG. 6 shows changes in skin-derived carbonyl oxide protein. At the time of formation, a decrease in water content, an increase in 8-OH dG, and an increase in carbonyl oxide protein were observed, but no significant changes were observed after 2 weeks of UV irradiation.

  The carbonyl oxide protein is a non-specific oxidized protein, and 8-OH dG is also non-specifically evaluating DNA oxidation, and “oxidation” is only one factor that causes wrinkles.

  On the other hand, genes extracted by comprehensive gene expression analysis are likely to be more closely related to the wrinkle formation process and are effective skin aging markers.

(Example 2)
Method for testing skin aging markers in cultured human cells

1. Culture of normal human epidermal keratinocytes (NHEK) NHEK (Cambrex Corporation) was added to serum-free basal medium (KBM, Cambrex Corporation) as a factor (0.1 ng / ml EGF, 0.03 mg / ml bovine vitality medium, 5 μg / ml) Insulin, 0.5 μg / ml hydrocortisone, GA-1000) was added to the medium KGM (+), and the cells were cultured in a 37 ° C., 5% CO ₂ incubator. Cells were seeded in 1 × 10 ⁵ cells / 60 mm dishes, the medium was changed every 2 days, and the cells were subcultured up to the fifth generation when they reached 80% saturation. Cells of the fifth generation were irradiated with ultraviolet B waves (UVB) at 0, 3 and 6 mJ / cm ² every 2 days, and were collected after culturing until 80% saturation.

2. Measurement of SA-β-Gal activity at each passage number of NHEK SA-β-Gal activity at each passage number of NHEK was measured using a Sensitive Cells Staining Kit (Sigma Corporation). Separately from subculture, cells are seeded in 3 dishes with 1 × 10 ⁵ cells / 60 mm dish, and at 80% saturation, cells are fixed in 2% formaldehyde / 0.2% glutaraldehyde aqueous solution at room temperature for 7 minutes. did. Further, after washing 3 times with 1 × PBS (−), staining solution was added and stained with over night at 37 ° C. For each of the three dishes, the number of cells stained with SA-β-Gal activity per 100 cells was counted, and an average value and a standard error were calculated.

3. Preparation of cell extract (CL) Wash the NHEK-saturated dish three times with PBS (-), add 1% SDS / 20 mM Tris-HCl (pH 7.5), and stir the protein for 30 minutes at 4 ° C. Extracted. Thereafter, the mixture was centrifuged at 15,000 × g for 30 minutes to remove insoluble matters, and the supernatant was used as a sample. Proteins were quantified by the Bradford method using DC protein assay kit (Bio-Rad Laboratories Inc.) and used as a sample for Western blotting.

4). Western Blotting SDS-PAGE was performed by Laemmli's Tris-Glycin system using samples for Western blotting. After SDS-PAGE, the protein in the gel was transferred to a PVDF membrane (Millipore Corporation) at a constant current of 0.8 mA per cm ² , soaked in 5% skim milk / PBS (−), and blocked at 4 ° C. and overnight. The PVDF membrane was washed 3 times with 0.1% Tween20 / PBS (−), then immersed in 5% skim milk / PBS (−) to which the primary antibody was added, and shaken at room temperature for 2 hours.
The types and dilution ratios of the primary antibodies used are as follows: mouse anti-β-catenin monoclonal antibody (clone 9G2, nanoTools Antitechtechntech GmbH & Co.) (1,000-fold dilution) A1 polyclonal antibody (Orbigen, Inc.) (1,000-fold dilution). As secondary antibodies, Horseradish peroxidase labeled anti-mouse IgG (Amersham Bioscience) (10,000-fold dilution), Horseradish peroxidase labeled anti-rabbit IgG (Amersham) was used. Next, it detected using Enhanced chemiluminescence (ECL) kit (Amersham Bioscience).
The expression level of the protein detected by Western blotting was quantified by NIH image, and the expression ratio was calculated with the expression level of the second generation protein as 1.

Experimental results Morphological changes associated with aging of epidermal keratinocytes Normal human cells stop proliferating after a certain number of divisions. This phenomenon is called cell senescence, but the number of divisions varies depending on the cell type. The human epidermal keratinocytes used in this experiment stop proliferating at about the 5th to 6th generations by repeated passages. In addition, the cell morphology in the fifth generation was about twice as large as that in the second generation, indicating a form of flattening observed by cell aging {FIG. 8 (b)}.
Moreover, there is a possibility of cell senescence or terminal differentiation as a cause of a decrease in proliferation of epidermal keratinocytes that have been repeatedly passaged. Therefore, in order to determine whether epidermal keratinocytes are actually senescent, it is known that they are not expressed in terminal differentiation, but are expressed only in cellular senescence. The activity of SA-β-Gal identified as a marker for aging was examined (Proc. Natl. Acad. Sci. USA, 92, 9363-9367, 1995). In the second generation, cells stained blue like SA-β-Gal were not seen, but in the fifth generation, the number of cells stained blue increased to about half (FIG. 9).
Based on the above results, cell senescence markers were searched using the second-generation cells as young cells, the third-generation and fourth-generation cells as transitional aging cells, and the fifth-generation cells as senescence cells.

2. Analysis of changes in expression of β-catenin and melanoma antigenic family A1 by aging of epidermal keratinocytes and UV irradiation by Western blotting β-catenin binds to DNA and other proteins, especially binding activity to cadherin, an adhesion molecule In addition to cell-cell adhesion, it is not limited to skin tissue as a major molecule of transcription factor activation, TGFβ signal pathway and Wnt / βcatenin signal pathway, and is involved in canceration of each tissue. For example, it is known that β-catenin is activated and enters the cell nucleus to switch on the proliferation of cancer cells, and C-Myc and the like increase via CRD-BP (coding region determinant-binding protein) (Nature 2006). ; 441 (7095): 898-901).

Melanoma antigen Family A family is expressed in cytotoxic T cells (CTL), and there are 12 adjacent molecular species. MAGE-A1 gene expression and secretory protein expression have been observed in melanoma (melanoma) cancer patients (Br J Dermatol. 2003; 149 (2): 282-8.).
For β-catenin and melanoma antigen family A1, Western blotting was performed using the respective antibodies in order to further confirm expression changes associated with aging and ultraviolet irradiation. As a result, the expression levels of β-catenin and melanoma antigen family A1 decreased with aging of keratinocytes and ultraviolet irradiation (FIGS. 10 and 11). This coincided with the fluctuation in the B1 and B4 gene expression in the mouse skin shown in Example 1. β-catenin and melanoma antigen family A1 are proteins derived from human skin B1 and B4 gene products, respectively.

Example 3
Skin aging marker verification test on protein samples collected from humans
Experimental method 1. Measurement of elasticity and wrinkle volume ratio of eye corners Elasticity and wrinkle volume ratio of eye corners were measured for 12 women in their 20s and 60s. The elasticity of the outer corner of the eye was measured by a cutometer (Courage + Khazaka electronic GmbH). Suction elasticity is a measurement method that serves as an index of skin elasticity, and it has been clarified that elasticity decreases with age. The volume ratio of the wrinkles of the corner of the eye was obtained by collecting a replica of the corner of the eye using a skin cast (Yamada Cosmetic Laboratories) which is a two-component mixed replica agent, and measuring the volume ratio of the wrinkle by replica image analysis. The replica image analysis was performed using a reflection replica analysis system ASA-03RXD. Using the ASA-03RXD, a shadow image corresponding to the shape of the wrinkles obtained by irradiating the sampled replica with parallel light at an angle of 30 degrees is captured by a CCD camera, captured in a computer, and processed for image processing. wrinkles volume fraction ^{(μm 3 / mm 2/100} ) was measured. It has been shown that the wrinkle volume ratio increases with aging.

2. Extraction of protein from the stratum corneum of the corner of the eye For the twelve women in their 20s and 60s, the stratum corneum is peeled from the corner of the eye using a stratum corneum checker (Asahibimed Co. Ltd.), and a protein extraction solution is used therefrom Extracted the protein. Specifically, a stratum corneum checker was affixed to the subject's eye corner and rubbed lightly with a fingertip. This was repeated 5 times. Thereafter, 50 μl of protein extraction solution {50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 0.4% Igepal CA-630 (Sigma-Aidrich Co.), respectively, on the stratum corneum attached to the five stratum corneum checkers. } And the stratum corneum was adapted to the protein extraction solution using a cell scraper, and then the solution was collected in a 1.5 ml plastic tube (Eppendorf AG).
Centrifugation was performed at 10,000 × g for 15 minutes to precipitate insoluble matter, and the supernatant was collected in a new 1.5 ml plastic tube. The amount of protein in the collected sample was measured using DC assay kit (Bio-Rad Laboratories, Inc.).

3. Measurement of skin aging marker The protein extracted from the corner of the corner of the eye corner was detected by ELISA (Enzyme-Linked Immunosorbent Assay) using the β-catenin antibody described in Example 2. Three micrograms of protein sample collected by tape stripping was coated on each well of the multiplate overnight at 4 ° C. Thereafter, the cells were blotted at 37 ° C. for 1 hour with 1% BSA / PBS, washed with PBS containing 0.1% Tween 20 three times, and reacted with β-catenin primary antibody diluted 1000 times at 37 ° C. for 1 hour. Again, the secondary antibody Horseradish peroxidase labeled anti-mouse IgG (Amersham Bioscience), which was washed 3 times with PBS containing 0.1% Tween 20 and diluted 10,000 times, was reacted at 37 ° C. for 1 hour. Thereafter, the plate was washed three times with PBS containing 0.1% Tween 20, and then a color reaction was performed using an Enhanced Chemiluminescence (ECL) kit (Amersham Bioscience), and then the chemiluminescence intensity was measured using the multiplate reader Lmax ^™ II384 (Molecular Devices Corporation.) And digitized with SoftMax® Pro installed in the same device.

Experimental Results Among the proteins whose expression changes with the aging of epidermal keratinocytes, it was examined whether or not β-catenin has a correlation with the degree of human skin aging.
As the index of human skin aging, the elasticity and wrinkle volume ratio of the outer corner of the eye, whose correlation with aging has been clarified, were used. In 12 women in their 20s to 60s whose skin aging degree was measured in this test, the elasticity of the corners of the eyes decreased significantly with age, and the wrinkle volume ratio increased significantly (FIGS. 12 and 13).
FIG. 14 shows the correlation between the expression level of β-catenin and the age in a protein sample collected from the vicinity of the corner of the eye by tape stripping. The expression level of β-catenin decreased with aging.
FIG. 15 shows the correlation between the expression level of β-catenin and the wrinkle volume ratio in the protein sample recovered from the vicinity of the corner of the eye by tape stripping. The expression level of β-catenin decreased significantly as the volume ratio of the corner of the eye corner increased.

  From the above results, it was revealed that β-catenin, a marker found using mouse skin gene expression analysis and cell aging of epidermal keratinocytes as an index, correlates with the degree of human skin aging.

The graph which shows the wrinkle improvement effect at the time of antioxidant intake. The graph which shows transition of a skin external appearance (wrinkle score). The graph which shows the change accompanying ultraviolet irradiation of skin moisture content. Image showing immunohistochemical staining of skin tissue 8-hydroxy-2'-deoxyguanosine. The graph which shows the change accompanying the ultraviolet irradiation of skin tissue 8-hydroxy-2'-deoxyguanosine. The graph which shows the change accompanying ultraviolet irradiation of skin origin carbonyl oxide protein. The gene expression path | route table image figure. The stained image of the cell which dye | stained the epidermis keratinocyte which irradiated the ultraviolet-ray B wave in each passage number and the 5th by SA- (beta) -Gal is shown. Epidermal keratinocytes that have been irradiated with ultraviolet B waves at passage 2 (a) and passage 5 (b-d) are stained with SA-β-Gal, and images of the stained cells are shown. The degree of cell senescence of epidermal keratinocytes in passage 2 to 5 and the degree of cell senescence of epidermal keratinocytes when irradiated with ultraviolet B waves are stained with SA-β-Gal and stained. The percentage of cells that were damaged. The result of having analyzed the expression change of (beta) -catenin accompanying senescence and ultraviolet irradiation of an epidermal keratinocyte is shown. The result of having analyzed the expression change of melanoma antigen family A1 accompanying the aging of an epidermal keratinocyte and ultraviolet irradiation is shown by the western blotting. The correlation diagram of age and skin elasticity in 12 women is shown. The correlation diagram of age and wrinkle volume ratio in 12 women is shown. The correlation diagram of age and expression level of β-catenin in 12 women is shown. The correlation diagram of wrinkle volume ratio and expression level of β-catenin in 12 women is shown.

Claims (3)

A test method for skin aging, which comprises measuring β- catenin.   The method for examining skin aging according to claim 1, wherein the skin aging is photoaging. The method according to claim 1 or 2, wherein β-catenin is measured by ELISA.