KR101045162B1 - Evaluation method of resistance to skin turning by ultraviolet rays - Google Patents

Abstract

The present invention expresses one or two or more proteins selected from the group consisting of annexin II, bleomycin hydrolase, cathepsin D, arginase-1, and SCCA2 in the skin stratum corneum obtained using non-invasive means. The present invention relates to a method for evaluating resistance to sunburn caused by ultraviolet rays using the amount as an index.

Description

Method for evaluating resistance to skin turning by ultraviolet light {Method for evaluating resistance to skin sunburn by ultraviolet light}

The present invention relates to a method for evaluating resistance to skin turnover by ultraviolet rays.

Sunburn occurs when the irradiated UV rays exceed melanin's protective capacity. There are two types of sunburn, which does not develop immediately after UV radiation, the skin becomes red after 2-6 hours, and the pain is most severe after 6 to 48 hours (sunburn), and 24-72 In time, there is a suntan where pigmentation proceeds. Sunburn refers to a condition in which UVB penetrates the epidermis and reaches the dermal papilla, resulting in hyperemia of capillaries in the papilla as an inflammatory reaction, resulting in redness of the color of the skin (erythema). When the amount of ultraviolet rays exceeds the defense reaction of the melanin pigment, cellular tissues are damaged, causing fever, blisters, and pain. This is called sun dermatitis. In addition, in tanning, UVA acts on melanocytes, promoting the production of melanin pigment. UVA does not involve redness and inflammation, but it causes melasma as it slows down the metabolism of the skin.

Ultraviolet rays, which cause sunburn, are concerned about many health effects such as carcinogenic effects on the skin, effects on eye diseases such as cataracts, and effects on immune function. Exposure to ultraviolet rays in the skin also causes various ultraviolet disorders. Ultraviolet rays can irritate the skin or oxidize unsaturated lipids in the skin to generate lipid radicals, producing lipid peroxide. Inflammation and oxidation caused by UV light cause tissue, cell, and DNA disorders. As a phenomenon by the ultraviolet exposure of the skin, wrinkles, blemishes, skin cancer, etc. are generally mentioned.

In recent years, there is also reported that the risk of skin cancer increases according to the difference in sensitivity to ultraviolet rays (skin color, skin type, etc.) as the dynamic data (Non-Patent Documents 1, 2 and 3). It is also clear that the risk of malignant melanoma (melanomer), which is a kind of skin cancer, increases several times due to the genetic variation of monobasic substitution of melanocortin1 receptor (Non-Patent Documents 4 and 5). Sensitivity to ultraviolet rays varies greatly depending on skin type (divided according to the number of sunburns and tanning conditions), divided into six types, or minimum UV energy that causes redness (erythema). (Minimum erythema: MED), but with the risk that the former cannot be measured without subjective observation and the latter with ultraviolet light. The increase in ultraviolet rays is more problematic than before due to the ozone layer destruction and the like, and the sensitivity risk to ultraviolet rays for an individual needs to be appropriately evaluated.

On the other hand, in the early stages of pigmentation, commonly called blemishes, epidermal keratinocytes are stimulated by various stimuli before they are present on the skin surface, such as α-MSH, bFGF, CGRP, and endothelin. Creates and transmits the information carrier to Melanosite. After delivery to melanocytes, a large amount of melanin pigment is made in the cells. In addition, turnover is abnormal due to ultraviolet rays, so the melanin pigment produced in large quantities cannot be discharged, and thus, the pigmentary lesion is changed. In the experiment using in vitro system, when the melanocytes were irradiated with UV, p73, Nup88, p27, Id1, and PCNA, which are proliferation factor markers of cells, also increased or decreased the expression of bcl-2, an apoptosis related protein. There is a report (Non Patent Literature 5).

Until now, there has been a report that the expression of NT-3, ADAM9, HB-EGF, etc. is markedly increased in the blemishes by comparing the blemishes with the normals by using biopsy skin tissue. (Patent Document 1). In addition, as a test method for predicting the formation of blemishes on the skin, it is reported that the amount of mRNA such as MLSTD1, MOGAT1, Mcp9, Krt2-6b, etc. is measured by biopsy to determine the risk of blemish formation (Patent Documents 2, 3, 4). ).

In addition, as a non-invasive method, the stratum corneum derived from skin collected through tape stripping or abrasion was used as a sample, and the amount of interleukin 1 (IL-1) and interleukin 1 receptor antagonist (IL-1ra) was analyzed and cortical. Patent Literature 5 discloses a method for evaluating.

Patent Document 1: Japanese Patent Application Laid-Open No. 2004-205246

Patent Document 2: Japanese Patent Application Laid-Open No. 2005-106745

Patent Document 3: Japanese Patent Application Laid-Open No. 2005-110505

Patent Document 4: Japanese Patent Application Laid-Open No. 2007-289063

Patent Document 5: Japanese Patent No. 3590708

[Non-Patent Document 1] Arch Dermatol. 2004; 140: 819-824

[Non-Patent Document 2] Cancer Causes Control. 1997; 8 (2): 246-252

Non-Patent Document 3: Ann Epidemiol. 2008; 18 (8): 614-627

Non-Patent Document 4: Invest Dermatol. 117: 294-300,2001

Non-Patent Document 5: Am. J. Hum. Genet. 66: 176-186, 2000

Non-Patent Document 6: Carcinogenesis 24 (12): 1929-1934,2003

An object of the present invention is to provide an index of resistance to skin turnover using a skin stratum corneum taken using non-invasive means. In addition, the resistance to skin change referred to in the present invention means that inflammation occurring within 24 hours after irradiation by ultraviolet exposure is recovered early and the degree of redness is reduced.

(1) Expression amount of one or two or more proteins selected from the group consisting of bleomycin hydrolase, cathepsin D, and arginase-1 in the stratum corneum of the skin A method for evaluating resistance to sunburn caused by ultraviolet light, characterized in that as an index.

(2) A method for selecting a skin anti-rotation cosmetic based on the evaluation method in (1).

In the present invention, the stratum corneum obtained by using a non-invasive technique, annexin II, bleomycin hydrolase, cathepsin D, arginase-1, squamous cell carcinoma-related antigen2 ( It was made clear that the amount of SCCA2) was indicative of resistance to skin turnover by ultraviolet rays.

According to the present invention, it is possible to evaluate the resistance to skin turning caused by ultraviolet rays without actually irradiating the ultraviolet rays.

Since the present invention can be evaluated using the skin stratum corneum collected using non-invasive tape stripping means, it is safe and simple.

In addition, the evaluation method of the present invention can be used to screen components useful for increasing resistance to skin turnover by ultraviolet rays.

According to the present invention, it is possible to evaluate the recovery ability of inflammation (erythema) by ultraviolet rays. If the recovery of inflammation caused by ultraviolet rays is low, it is estimated that the risk of skin aging, wrinkles, blemishes, and even skin cancer caused by UV exposure is high. Beauty counseling, such as recommending the use of a screen agent, can be performed accurately.

EMBODIMENT OF THE INVENTION Hereinafter, embodiment of this invention is described in detail.

This invention can know the resistance to the skin change by the ultraviolet-ray of the said subject based on the abundance which measured the 5 types of proteins which exist in the skin stratum corneum collected by the non-invasive method. The present invention was completed by discovering a correlation between the degree of recovery after exposure to ultraviolet rays and the abundance of the five kinds of proteins. These five types of proteins are annexin II, bleomycin hydrolase, cathepsin D, arginase-1, and SCCA2. Annexin II, bleomycin hydrolase, and cathepsin D tend to have a strong resistance to skin bleeding due to ultraviolet rays in people with little protein expression. Arginase-1 and SCCA2 have a tendency to have a strong resistance to skin bleeding due to ultraviolet rays in those with a large amount of protein expression.

Therefore, for these five types, the expression level can be set in several steps in advance, and the degree of skin turning resistance by ultraviolet rays can be indexed. By measuring the expression amount of the skin stratum corneum taken from the subject and comparing it with these indices, the skin turning resistance of the subject can be easily known.

Based on these results, you can direct those who need attention to sunburn to watch out for outdoor activities. Or when it turns out that it is weak to an ultraviolet-ray, each person can take measures, such as selecting cosmetics with a strong ultraviolet shielding effect. Alternatively, the counseling of cosmetic selection can be conducted on a scientific basis.

In addition, by using a cultured skin model that is susceptible to UV radiation, screening for substances that affect the expression levels of these five types of proteins, screening for drugs that can improve UV resistance by UV irradiation It becomes possible.

In the present invention, the skin stratum corneum can be safely and easily collected by a non-invasive method such as tape stripping, and the expression level of the protein of interest in a short time can be specified using the skin stratum corneum. For this reason, it can measure easily in a cosmetics store etc. and can utilize it as information of cosmetic selection on the spot. The instrument for measuring the expression level of this protein can be presented by comparing the detected result with the staged index. Moreover, based on this result, it is also possible to display suitable cosmetics.

Moreover, this invention can evaluate aging risks, such as skin aging, for the purpose other than a medical purpose, and can take countermeasures for the purpose other than medical care based on it.

Arginase-1 is an intracellular protein with a molecular mass of 34,735 Da, an enzyme that converts arginine to ornithine and urea. There are two or more isoforms (types I and II) of mammalian arginase, and they differ in tissue distribution, intracellular locality, immunological cross-reactivity, and physiological function. The isoform type I, commanded by this gene, is a cytosol enzyme and is primarily expressed in the liver as a constituent of the urea cycle. The genetic "defect" of this enzyme results in "arginineemia," an autosomal recessive hereditary disease characterized by "hyperammonemia." Gene sequencing information (ARG1, Nucleic Acids Res.:16:8789-8802 (1988), X12662). Amino acid sequence information (Arginase-1, Cell Death Differ. 2000 Feb; 7 (2): 137-44, P05089).

Annexin II is an intracellular protein with a molecular mass of 38,473 Da, which is a membrane-bound protein controlled by calcium, and two calcium ions bind to this protein. Of the two groups of annexin repeats, one binds calcium and one binds phospholipids. The protein crosslinks with actin or cytoskeleton proteins that bind to the phospholipids in the cell membrane, or activates plasminogen via a tissue plasminogen activator (t-PA). Gene sequencing information (Annexin A2, Gene 95: 243-251 (1990), BC015834). Amino acid sequence information (Annexin A2, J. Biol. Chem. 266: 5169-5176 (1991), P07355).

Bleomycin hydrolase is an intracellular protein with a molecular mass of 52,562 Da, one of the neutral cysteine proteases of the papain family. The only known activity is metabolic inactivation of glycopeptide bleomycin (BLM) (an important component of cancer chemotherapy). Gene sequencing information (BLMH, 1230091253543_0, X92106). Amino acid sequence information (bleomycin hydrolase, Biochemistry 35: 6706-6714 (1996), Q13867).

Cathepsin D is a lysosomal aspartic acid protease with a molecular mass of 44,542 Da, which promotes the breakdown of intracellular proteins. It has been suggested to be involved in the onset of inflammation, atherosclerosis, thrombosis, Alzheimer's disease, and the like, and also involved in the proliferation of tumor cells such as apoptosis and breast cancer. Gene sequencing information (CTSD, 1230091253543_1, M63138). Amino acid sequence information (Cathepsin D, Nat. Biotechnol. 21: 660-666 (2003), P07339).

Squamous cell carcinoma-related antigen squamous cell carcinoma-related antigen2 (SCCA2) is an intracellular protein with a molecular mass of 44,854 Da, a serine protease inhibitor having a serpin structure at the carboxyl terminal side. The expression is small in normal squamous epithelium, but high in squamous cell carcinoma. In addition to strongly inhibiting cathepsin G and serine proteases of chymases, cysteine protease, papain, and cathepsin L are also inhibited. Gene sequencing information (SERPINB4, 1230091253543_2, X89015). Amino acid sequence information (Serpin B4, Int. J. Cancer 89: 368-377 (2000), P07355).

The arginase-1, annexin II, bleomycin hydrolase, cathepsin D, and SCCA2 may be precursor proteins, mature proteins, or may be cleaved or non-cleaved. Examples of the precursor protein include proproteins, preproproteins, and the like. Some proproteins, preproproteins, and the like have a signal peptide.

How to collect stratum corneum proteins from the stratum corneum

(1) Collection of stratum corneum by tape stripping method

The tape stripping method is a method of collecting a stratum corneum adhered to an adhesive face by applying an adhesive tape to the skin and peeling off the adhesive tape.

By using a commercially available adhesive tape, for example, the adhesive tape is pressed onto the human narrow part, the stratum corneum layer can be adhered to the adhesive tape and collected. Commercially available adhesive tapes include keratin checkers made by Asahi Biomed, keratin layer yarns made by Mortex, keratin checkers made by PROMOTOOL (disk type W, disc type G, PRO type), Corneofix made by Integral, transparent tape made by 3M, transparent double-sided tape made by 3M, etc. Can be mentioned.

(2) extraction buffer

An extraction buffer is used to extract the stratum corneum protein from the stratum corneum adhered to the adhesive tape. As an extraction buffer, the following compositions are mentioned, for example. Composition: PBS (-), 0.1% SDS.

The stratum corneum of the skin, which is collected by the adhesive tape, is differentiated from keratinocytes and contains many proteins of the cytoskeletal system. Therefore, in order to raise the extraction efficiency of a stratum corneum protein, it is preferable to contain a suitable surfactant in this extraction buffer. It is preferable to use SDS (sodium lauryl sulfate, CH ₃ (CH ₂ ) ₁₁ OSO ₃ Na) as the surfactant.

(3) extraction of stratum corneum protein

The adhesive tape from which the stratum corneum is collected is placed in a cylindrical container, and the stratum corneum protein can be quickly extracted by rubbing the adhesive surface using a pestle for homogenization (hereinafter referred to as "pestle") which rotates at high speed. The adhesive tape faces the adhesive face inside the cylindrical container, and puts the opposite side of the adhesive face along the inside of the cylindrical container, and puts it in the cylindrical container. By putting the adhesive tape on the opposite side of the adhesive surface along the inside of the cylindrical container, the adhesive surface can be easily rubbed with the pestle. As another method, the adhesive tape from which the stratum corneum is taken may be immersed in an extraction buffer and rubbed with a scraper to extract the stratum corneum protein, or a shaking method or an ultrasonic irradiation method may be used.

(4) Recovery of the horny layer protein extract

It is preferable that the stratum corneum protein extract is left to stand for about 1 minute, the bubbles are precipitated, and thereafter, the stratum corneum protein extract is sucked and recovered from the cylindrical container.

(5) Analysis of abundance of arginase-1, annexin II, bleomycin hydrolase, cathepsin D, SCCA2 in stratum corneum protein

The amount of arginase-1, annexin II, bleomycin hydrolase, cathepsin D and SCCA2 in the obtained stratum corneum protein extract can be quantitatively analyzed by immunoblotting, ELISA, antibody chip or FRET. .

UV skin In advance  Evaluation of resistance to

It became clear that the amount of annexin II, bleomycin hydrolase, and cathepsin D of the group in which skin erythema disappears after 1 week of ultraviolet irradiation of 1 MED is small compared with the group which does not lose skin number.

Moreover, it became clear that the amount of arginase-1 and SCCA2 of the group which loses skin number after 1 week of ultraviolet irradiation of 1 MED compared with the group which does not lose skin number.

Therefore, when the amount of annexin II, bleomycin hydrolase, and cathepsin D in the stratum corneum is less than the mean value, or when the amount of arginase-1 and SCCA2 is greater than the mean value, It can be said that the resistance to this is strong.

Conversely, those whose amounts of annexin II, bleomycin hydrolase, and cathepsin D in the stratum corneum are higher than the average value, or those whose amounts of arginase-1 and SCCA2 are lower than the average value are aged by skin exposure. It can be evaluated that blemishes and wrinkles tend to be enhanced, and UV disturbances such as blemishes are likely to occur.

UV skin Forward  Screening of Dissipation Accelerators

By reducing the amount of annexin II, bleomycin hydrolase and cathepsin D in the stratum corneum of the mouse or the like, or searching for a component that increases the amount of arginase-1 and SCCA2, Screening promoters for elimination of skin turnover can be screened.

Example

1. Subject

Ten males in their twenties and thirties were enrolled. Table 1 shows the age, skin type, minimum erythema amount, and a * value before ultraviolet irradiation of each subject. In addition, skin type II is a type of `` simple sunburn, slightly tanning '', and skin type III is `` normally sunburn, usually tanning '' (skin tan). IV is a type that causes a slight sunburn and always tans (brown tan). The skin types shown in Table 1 are self-reported by the subject. Therefore, the minimum amount of erythema measured in this objective and skin type does not necessarily correspond.

In addition, a * value was measured as an index of the redness level which arises with inflammation by an ultraviolet-ray.

2. Determination of the amount of arginase-1, annexin II, bleomycin hydrolase, cathepsin D, and SCCA2 in the stratum corneum

2.1 Collection of the stratum corneum by tape stripping

Asahi Biomed Co., Ltd. keratin checker (2.5 cm x 2.5 cm) was used as the adhesive tape. A keratin checker was attached to the subject's back, lightly rubbed with a fingertip, and the skin stratum corneum was adhered to the adhesive surface of the keratin checker, and a total of five tape strips were taken from the back. In addition, the sampling place by tape stripping was extract | collected from the ultraviolet non-irradiation site | part.

2.2 Extraction of Skin Corneal Proteins

Extraction buffer: PBS (-), 0.1% (w / v) SDS.

Extraction buffer volume: 200 μL.

Extraction vessel: A cylindrical vessel having a diameter of 11 mm.

Put the adhesive tape into the cylindrical container so that the opposite side of the adhesive surface is along the inner wall of the container, and put the extraction buffer, and put Handy pestle (TOYOBO company, code HMX-301) as a pestle to a mini cordless grinder ), The pestle was rotated at 9000 rpm, and the adhesive surface of the adhesive tape which followed the cylindrical container inner wall was rubbed. The stirring time was 90 seconds.

The skin stratum corneum protein extract was left to stand for about 1 minute, the foam was allowed to settle, and then the skin stratum corneum protein extract was aspirated and recovered from the cylindrical container.

3. Measurement of the amount of protein of the stratum corneum of the skin

The total protein contained in the stratum corneum protein extract was measured using a DC protein assay kit (BIO-RAD).

4. Determination of the amount of arginase-1, annexin II, bleomycin hydrolase, cathepsin D, and SCCA2 in the stratum corneum protein

The amount of expression of arginase-1, annexin II, bleomycin hydrolase, cathepsin D, and SCCA2 by immunoblotting was measured using an amount of 1 µg of skin stratum corneum protein. After separating by SDS-PAGE using a 5-15% gradient gel using 1 µg of skin stratum corneum protein of each subject, the protein was transferred to PVDF membrane and blocked with 5% skim milk. Primary antibody (Arginase-1 monoclonal antibody: Santa Cruz Biotechnology, Annexin II polyclonal antibody: Santa Cruz Biotechnology, Bleomycin hydrolase polyclonal antibody: Abnova, cathepsin D monoclonal antibody: R & D Systems, SCCA2 Monoclonal antibody: Santa Cruz Biotechnology) was diluted 1000-fold, secondary antibody (HRP-mouse anti-Goat IgG: ZYMED Laboratories, HRP-rabbit anti-Goat IgG: ZYMED Laboratories) at 10000-fold dilution, and then ECLplus ( BD Bioscience). The intensity of the obtained band was digitized by NIH-Image. This value was taken as the amount of arginase-1, annexin II, bleomycin hydrolase, cathepsin D, and SCCA2.

5. UV irradiation

The subject's back was irradiated with 1 MED ultraviolet light in a circle of about 8 mm in diameter. The UV irradiation apparatus used Multiple Solar Ultraviolet Simulator Model 601 made by solar light company. UV intensity was set using Erythema UV & UVA Intensity Meter Model 3D-600 v2.0 manufactured by solar light company.

6. Measurement of skin a ＊ value

In order to evaluate the degree of erythema of skin sunburn, the a * value of the ultraviolet irradiation site and the non-irradiation site | part was measured using the spectrophotometer (CM-500, the product made by Konica Minolta). Measure the a * value of the UV irradiation site and the non-irradiation site on 2, 4, 7, 10, and 14 days after irradiation, and measure the difference of a * between the irradiation site and the non-irradiation site within the same day. It was used for analysis as an a * change amount (Δa *) of.

7. Measurement result of skin a ＊ value

The change amount (Δa *) of the a * value after ultraviolet irradiation is shown in FIG. 1, and the Δa * value on the 7th day is shown in Table 2. FIG.

Skin erythema with Δa * as an index reaches a maximum in 24 hours after irradiation, and this decreases over time. And on day 7, it reaches about 1/4 of its maximum value.

Subjects 1 to 5 almost lost erythema on the 7th day of ultraviolet irradiation (Δa * <1), while subjects 6 to 10 did not lose erythema on the 7th day of ultraviolet irradiation (Δa ＊ ＞ 1). If Δa *> 1, erythema can be confirmed by visual evaluation, and all subjects 6-10 confirmed erythema by visual evaluation. On the other hand, erythema of test subjects 1-5 was not confirmed by visual evaluation.

Accordingly, the subjects 1 to 5 have a faster recovery of redness than the subjects 6 to 10, and can be evaluated as having high resistance to skin turnover.

Resistance to the skin change of the present invention indicates the superiority of the function that the minimum erythema is lost and inflammation is restored when the degree of skin change is unified to the minimum erythema. Therefore, the minimum erythema amount of ultraviolet rays and the resistance to skin turning do not necessarily correlate.

The skin types shown in Table 2 are classified by questionnaire survey of the subject's own sunburn to the last, and there is a part which does not match the minimum erythema amount in Table 2, but this is generally classified by oneself The skin type that is present has many ambiguities, and the minimum amount of erythema and the protein expression measured this time may indicate more objective indices.

8. Amounts of Arginase-1, Annexin II, Bleomycin Hydrolase, Cathepsin D, and SCCA2 in the Skin No. Loss and UV No.

7. After 7 days of ultraviolet irradiation shown in Table 2, divided into two groups of the group where the skin number was lost (subjects 1 to 5) and the group where the skin number was not lost (subject 6 to 10), and the subjects of each group The average values of the amount of arginase-1, annexin II, bleomycin hydrolase, cathepsin D, and SCCA2 in the stratum corneum of the skin were compared. The results are shown in FIGS. 2 to 6.

As shown to FIG. 2, FIG. 6, the arginase-1 abundance average value of the erythema arsenic group was 2.166, but the arginase-1 abundance average value of the erythema disappearance group was 8.081, 3.73 times. There was a significant difference in the mean value of arginase-1 in the erythema non-depleted group and the erythema depleted group (p = 0.038). In addition, while the mean value of SCCA2 abundance in the erythema non-loss group was 4.220, the mean value of SCCA2 abundance in the erythema disappearance group was 12.377, and a difference of 2.93 times was confirmed.

People with high amounts of arginase-1 and SCCA2 present in the stratum corneum were evaluated as having high resistance to skin turnover.

3 to 5, the mean value of Annexin II abundance in the erythema non-loss group was 2.598, whereas the mean value of Annexin II abundance in the erythema disappearance group was 1.286, which was 0.49 times. The mean value of Annexin II abundance was significantly different in the erythema no shedding group and the erythema loss group (p = 0.011). In addition, the mean value of bleomycin hydrolase abundance in the erythema non-loss group was 13.756, whereas the mean value of bleomycin hydrolase abundance in the erythema disappearance group was 4.961, and 0.36-fold difference was confirmed. The mean value of cathepsin D abundance in the erythema non-loss group was 27.757, whereas the mean value of cathepsin D abundance in the erythema disappearance group was 8.705, confirming a 0.31-fold difference.

People with high amounts of annexin II, bleomycin hydrolase, and cathepsin D in the stratum corneum were evaluated to have low resistance to skin turnover.

Brief description of the drawings

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the amount of change of the a * value after ultraviolet irradiation.

2 is a graph comparing the average value of the amount of arginase-1 present in the stratum corneum of the subject.

3 is a graph comparing average values of the amount of Annexin II present in the stratum corneum of the subject.

4 is a graph comparing the average value of the amount of bleomycin hydrolase present in the stratum corneum of the subject.

5 is a graph comparing the average value of the amount of cathepsin D present in the stratum corneum of the subject.

6 is a graph comparing average values of the amount of SCCA2 present in the stratum corneum of the subject.

Claims (2)

Skin sunburn by ultraviolet light, characterized by the expression level of one or two or more proteins selected from the group consisting of bleomycin hydrolase, cathepsin D, and arginase-1 in the stratum corneum of the skin Method for evaluating resistance to A method for selecting a skin anti-rotation cosmetic based on the evaluation method of claim 1.

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