JP2012039970A - Method for examining degree of skin damage - Google Patents
Method for examining degree of skin damage Download PDFInfo
- Publication number
- JP2012039970A JP2012039970A JP2010185680A JP2010185680A JP2012039970A JP 2012039970 A JP2012039970 A JP 2012039970A JP 2010185680 A JP2010185680 A JP 2010185680A JP 2010185680 A JP2010185680 A JP 2010185680A JP 2012039970 A JP2012039970 A JP 2012039970A
- Authority
- JP
- Japan
- Prior art keywords
- skin
- expression
- skin damage
- stratifin
- exposure
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 59
- 230000037380 skin damage Effects 0.000 title claims abstract description 56
- 230000014509 gene expression Effects 0.000 claims abstract description 82
- 102100027833 14-3-3 protein sigma Human genes 0.000 claims abstract description 57
- 108050008974 14-3-3 protein sigma Proteins 0.000 claims abstract description 55
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 40
- 102000011782 Keratins Human genes 0.000 claims description 22
- 108010076876 Keratins Proteins 0.000 claims description 22
- 238000012360 testing method Methods 0.000 claims description 9
- 238000003752 polymerase chain reaction Methods 0.000 claims description 3
- 108020004999 messenger RNA Proteins 0.000 abstract description 40
- 239000002537 cosmetic Substances 0.000 abstract description 10
- 210000003491 skin Anatomy 0.000 description 48
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 19
- 239000000523 sample Substances 0.000 description 11
- 108010085238 Actins Proteins 0.000 description 9
- 102000007469 Actins Human genes 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 238000010998 test method Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 210000002510 keratinocyte Anatomy 0.000 description 6
- 238000011002 quantification Methods 0.000 description 6
- 230000006378 damage Effects 0.000 description 5
- 210000001339 epidermal cell Anatomy 0.000 description 5
- 230000004224 protection Effects 0.000 description 5
- 210000000434 stratum corneum Anatomy 0.000 description 5
- 230000000475 sunscreen effect Effects 0.000 description 5
- 239000000516 sunscreening agent Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 206010042496 Sunburn Diseases 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000019612 pigmentation Effects 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 230000037303 wrinkles Effects 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 208000000453 Skin Neoplasms Diseases 0.000 description 3
- 239000000853 adhesive Substances 0.000 description 3
- 230000001070 adhesive effect Effects 0.000 description 3
- 239000002390 adhesive tape Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 230000003211 malignant effect Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 201000000849 skin cancer Diseases 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 101000723509 Homo sapiens 14-3-3 protein sigma Proteins 0.000 description 2
- 101000701902 Homo sapiens Serpin B4 Proteins 0.000 description 2
- 206010051246 Photodermatosis Diseases 0.000 description 2
- 102100030326 Serpin B4 Human genes 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000011382 collagen catabolic process Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 231100000040 eye damage Toxicity 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000008845 photoaging Effects 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 210000004927 skin cell Anatomy 0.000 description 2
- 108010088201 squamous cell carcinoma-related antigen Proteins 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 102000004149 Annexin A2 Human genes 0.000 description 1
- 108090000668 Annexin A2 Proteins 0.000 description 1
- 102100021723 Arginase-1 Human genes 0.000 description 1
- 101710129000 Arginase-1 Proteins 0.000 description 1
- 102100027058 Bleomycin hydrolase Human genes 0.000 description 1
- 108010025544 Bleomycin hydrolase Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000002177 Cataract Diseases 0.000 description 1
- 102000003908 Cathepsin D Human genes 0.000 description 1
- 108090000258 Cathepsin D Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 208000034656 Contusions Diseases 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000642478 Homo sapiens Serpin B3 Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102000005706 Keratin-6 Human genes 0.000 description 1
- 108010070557 Keratin-6 Proteins 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 208000012641 Pigmentation disease Diseases 0.000 description 1
- 201000002154 Pterygium Diseases 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 1
- 102100036383 Serpin B3 Human genes 0.000 description 1
- 206010040954 Skin wrinkling Diseases 0.000 description 1
- 230000006750 UV protection Effects 0.000 description 1
- 238000005299 abrasion Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 208000009621 actinic keratosis Diseases 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 210000001217 buttock Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000006369 cell cycle progression Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 210000000245 forearm Anatomy 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 206010023332 keratitis Diseases 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 239000004745 nonwoven fabric Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- LOIYMIARKYCTBW-OWOJBTEDSA-N trans-urocanic acid Chemical compound OC(=O)\C=C\C1=CNC=N1 LOIYMIARKYCTBW-OWOJBTEDSA-N 0.000 description 1
- LOIYMIARKYCTBW-UHFFFAOYSA-N trans-urocanic acid Natural products OC(=O)C=CC1=CNC=N1 LOIYMIARKYCTBW-UHFFFAOYSA-N 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Abstract
Description
本発明は、皮膚のｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子の発現量を測定することを特徴とする、紫外線曝露による皮膚損傷の程度を検査する方法に関する。 The present invention relates to a method for examining the degree of skin damage due to ultraviolet exposure, which comprises measuring the expression level of a gene encoding a stratifin protein in skin.
紫外線は生物学的観点から、ＵＶＡ（長波長域、４００〜３１５ｎｍ）、ＵＶＢ（中波長域、３１５〜２８０ｎｍ）、ＵＶＣ（短波長域、２８０ｎｍ未満）に分類される。人体への過度の紫外線曝露は、主に眼損傷と皮膚損傷を引き起こす。紫外線曝露による眼損傷の症状として、角膜炎、翼状片、白内障等が知られている。又、紫外線曝露による皮膚損傷の症状として、日焼け、色素沈着、シワ・タルミ、良性腫瘍、前がん症（日光角化症、悪性黒子）、皮膚がん等が知られている。紫外線曝露による皮膚損傷は、太陽光に曝露される皮膚である顔面又は首筋において起こりやすい。近年では、特にオゾン層の破壊進行により、紫外線曝露による皮膚損傷は問題となっている。 From a biological point of view, ultraviolet rays are classified into UVA (long wavelength range, 400 to 315 nm), UVB (medium wavelength range, 315 to 280 nm), and UVC (short wavelength range, less than 280 nm). Excessive UV exposure to the human body mainly causes eye damage and skin damage. Keratitis, pterygium, cataract, etc. are known as symptoms of eye damage due to exposure to ultraviolet rays. Further, as a symptom of skin damage due to exposure to ultraviolet rays, sunburn, pigmentation, wrinkle/tarumi, benign tumor, precancerous disease (keratokeratosis, malignant lentis), skin cancer and the like are known. Skin damage due to UV exposure is likely to occur in the skin exposed to sunlight, the face or the nape of the neck. In recent years, skin damage due to exposure to ultraviolet rays has become a problem particularly due to the progress of destruction of the ozone layer.
紫外線曝露後の反応には、個人差がある。少量の紫外線曝露により皮膚損傷を引き起こすヒトもいれば、比較的紫外線曝露に抵抗力のあるヒトもいる。このような紫外線曝露による皮膚損傷の程度の差は、遺伝や生活環境等の様々な要因が考えられるため、原因を特定することは難しい。しかしながら、紫外線曝露による皮膚損傷の程度を知ることができれば、どの化粧品を使用して皮膚損傷を予防したら良いか、或いは、どの化粧品を使用して紫外線曝露後の皮膚損傷をケアしたら良いか等が分かる。例えば、紫外線曝露により皮膚損傷を受けやすい肌状態であるという検査結果であれば、予め、日焼け止め化粧品を塗布したり、紫外線曝露後も、念入りに美白やアンチエイジング対応の化粧品を使用したりすることにより、色素沈着やシワ・タルミ等の皮膚損傷を予防・改善することができる。 There are individual differences in the response after UV exposure. Some humans cause skin damage with small doses of UV exposure, while others are relatively resistant to UV exposure. It is difficult to identify the cause of the difference in the degree of skin damage due to the exposure to ultraviolet rays because various factors such as genetics and living environment are considered. However, if it is possible to know the extent of skin damage due to UV exposure, what cosmetics should be used to prevent skin damage, or what cosmetics should be used to care for skin damage after UV exposure, etc. I understand. For example, if the test result shows that your skin is susceptible to skin damage due to UV exposure, you may apply sunscreen cosmetics in advance, or carefully use whitening and anti-aging cosmetics after UV exposure. By doing so, skin damage such as pigmentation and wrinkles and tarmi can be prevented and improved.
今までに、紫外線曝露による皮膚損傷の程度を検査する方法がいくつか発明されている。例えば、アポトーシスを抑制する扁平上皮がん関連抗原ＳＣＣＡ（Ｓｑｕａｍｏｕｓ Ｃｅｌｌ Ｃａｒｃｉｎｏｍａ Ａｎｔｉｇｅｎ）の一種であるＳＣＣＡ−１及び／又はＳＣＣＡ−２の発現を測定し、表皮の紫外線抵抗力を判断する方法（特許文献１、非特許文献１）、皮膚の近赤外吸収スペクトルから、紫外線曝露による損傷の度合いを判定する方法（特許文献２）、紫外線に曝露されることにより変化した角層内のウロカニン酸量をラマン分光法により測定する方法（特許文献３、非特許文献２）、及び、皮膚角層中のアネキシンＩＩ、ブレオマイシン ハイドロラーゼ、カテプシンＤ、アルギナーゼ−１、ＳＣＣＡ−２からなる群から選ばれる一種又は二種以上のタンパク質の発現量を指標にして、紫外線による皮膚サンバーンに対する抵抗性を評価する方法（特許文献４）等が知られている。しかしながら、実際のヒトの皮膚から有棘層及び顆粒層を含めた表皮細胞を採取する必要がある、複雑な測定機器を使用しなければならない、標的の因子自体がよく分かっていない、一部の皮膚損傷しか検査できない等、容易に紫外線曝露による皮膚損傷の程度を検査することは難しかった。 To date, several methods have been invented for examining the extent of skin damage due to UV exposure. For example, a method of determining the ultraviolet resistance of the epidermis by measuring the expression of SCCA-1 and/or SCCA-2, which is a type of squamous cell carcinoma-associated antigen SCCA (Squamous Cell Carcinoma Antigen) that suppresses apoptosis (Patent Document 1, non-patent document 1), a method for determining the degree of damage due to ultraviolet exposure from the near infrared absorption spectrum of the skin (patent document 2), and the amount of urocanic acid in the stratum corneum changed by exposure to ultraviolet light. A method of measuring by Raman spectroscopy (patent document 3, non-patent document 2), and one selected from the group consisting of annexin II, bleomycin hydrolase, cathepsin D, arginase-1, SCCA-2 in the stratum corneum of the skin, or A method of evaluating the resistance to skin sunburn caused by ultraviolet rays using the expression levels of two or more proteins as an index (Patent Document 4) and the like are known. However, it is necessary to collect epidermal cells including the spinous layer and the granular layer from actual human skin, complicated measurement equipment must be used, the target factor itself is not well understood, and some It was difficult to easily inspect the degree of skin damage due to UV exposure, such as the fact that only skin damage could be inspected.
本発明は、紫外線曝露によるヒトの皮膚損傷の程度を簡便に検査する方法を提供することを課題とする。 An object of the present invention is to provide a method for simply examining the degree of human skin damage due to exposure to ultraviolet rays.
本発明者は、ｓｔｒａｔｉｆｉｎに注目して、紫外線曝露によるヒトの皮膚損傷の程度を簡便に検査する方法について、鋭意検討した。その結果、ｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子の発現を調べることにより、皮膚損傷の程度を検査できることを見出した。又、この遺伝子の発現産物は、テープストリップ等によりヒトの皮膚から容易に、且つ、非侵襲的に採取することができることを見出した。 The present inventors paid attention to stratifin, and earnestly studied a method for simply examining the degree of human skin damage due to exposure to ultraviolet rays. As a result, they have found that the degree of skin damage can be examined by examining the expression of the gene encoding the stratifin protein. It was also found that the expression product of this gene can be easily and non-invasively collected from human skin by means of a tape strip or the like.
本発明は、以下に列挙する皮膚検査方法を提供する。
［１］皮膚のｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子の発現量を測定することを特徴とする、皮膚損傷の程度を検査する方法。
［２］前記皮膚損傷が紫外線曝露による皮膚損傷である、［１］に記載の方法。
［３］皮膚のｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子の発現量を基に、紫外線曝露による皮膚損傷の程度を検査する方法であって、
（１）被験対象から皮膚のサンプルを得る工程、
（２）該サンプルのｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子の発現を定量化する工程、
（３）工程（２）にて測定した遺伝子発現量を用いて、紫外線曝露による皮膚損傷の程度を検査する工程
を含む方法。
［４］前記遺伝子発現の定量化がポリメラーゼ連鎖反応法により行われる、［３］に記載の皮膚検査方法。
［５］前記皮膚サンプルが被験対象の皮膚から採取した角質である、［３］〜［４］のいずれかに記載の皮膚検査方法。
The present invention provides the skin test methods listed below.
[1] A method for inspecting the degree of skin damage, which comprises measuring the expression level of a gene encoding a stratifin protein in the skin.
[2] The method according to [1], wherein the skin damage is skin damage due to exposure to ultraviolet light.
[3] A method for inspecting the degree of skin damage due to UV exposure, based on the expression level of a gene encoding the stratifin protein of the skin,
(1) obtaining a skin sample from a test subject,
(2) quantifying the expression of a gene encoding the stratifin protein in the sample,
(3) A method including a step of inspecting the degree of skin damage due to UV exposure using the gene expression level measured in step (2).
[4] The skin test method according to [3], wherein the gene expression is quantified by a polymerase chain reaction method.
[5] The skin test method according to any one of [3] to [4], wherein the skin sample is keratin collected from the skin of a test subject.
本発明の方法により、皮膚のｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子の発現産物であるｍＲＮＡの発現量を測定し、その結果から、紫外線曝露による皮膚損傷の程度を検査するための方法を提供することが可能になる。 According to the method of the present invention, it is possible to provide a method for measuring the expression level of mRNA, which is an expression product of a gene encoding a stratifin protein in the skin, and examining the degree of skin damage due to ultraviolet exposure from the result. become.
本発明は、皮膚のｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子の発現産物であるｍＲＮＡの発現量を測定し、その結果から、紫外線曝露による皮膚損傷の程度を検査するための方法（以下、「皮膚損傷検査法」と言う）を提供する。本発明の皮膚損傷検査法では、被験対象の皮膚から採取したサンプルを用いて、ｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子発現の定量化を行う。その結果を基に、紫外線曝露により皮膚損傷を受けやすい肌状態であるのか否かを評価する。更に、皮膚から採取したサンプルとして角質を用いることにより、非侵襲的な評価が可能になる。 The present invention is a method for measuring the expression level of mRNA, which is an expression product of a gene encoding a stratifin protein in the skin, and examining the degree of skin damage due to ultraviolet exposure from the result (hereinafter, referred to as “skin damage test method”). Say). In the skin damage test method of the present invention, the expression of a gene encoding a stratifin protein is quantified using a sample collected from the skin of a test subject. Based on the result, it is evaluated whether or not the skin condition is susceptible to skin damage due to exposure to ultraviolet rays. Furthermore, by using keratin as a sample collected from the skin, non-invasive evaluation becomes possible.
本発明の「ｓｔｒａｔｉｆｉｎ（ＳＦＮ、別名１４−３−３σ）」とは、表皮細胞にて産生されるタンパクの一種であり、表皮細胞における細胞周期の進行を停止させたり、アポトーシスを誘導したりする作用を持つ（Ｂｈａｔｉａ Ｋ．，Ｃａｎｃｅｒ Ｅｐｉｄｅｍｉｏｌ．Ｂｉｏｍａｒｋｅｒｓ Ｐｒｅｖ．，１２，１６５−１６９，２００３．）。又、最近の知見から、真皮細胞にｓｔｒａｔｉｆｉｎタンパクを曝露させると、コラーゲン分解を亢進することも分かってきた（Ｇｈａｈａｒｙ Ａ．，Ｊ．Ｉｎｖｅｓｔ．Ｄｅｒｍａｔｏｌ．，１２２，１１８８−１１９７，２００４．）。 The “stratifin (SFN, also known as 14-3-3σ)” of the present invention is a kind of protein produced in epidermal cells, and stops the cell cycle progression in epidermal cells or induces apoptosis. It has an action (Bhatia K., Cancer Epidemio. Biomarkers Prev., 12, 165-169, 2003.). Further, recent findings have revealed that exposure of dermal cells to the stratifin protein promotes collagen degradation (Ghahary A., J. Invest. Dermatol., 122, 1188-1197, 2004.).
本発明者は、表皮細胞に紫外線を曝露させたところ、ｓｔｒａｔｉｆｉｎ ｍＲＮＡ発現量及びタンパク量が増加することを明らかにした。又、ｓｔｒａｔｉｆｉｎの発現は、非露光部位の上腕内側と比較して露光部位の顔面において亢進していることも明らかにした。これらの結果から、紫外線曝露によりｓｔｒａｔｉｆｉｎが増加し、表皮細胞における細胞周期の進行を停止させるために、表皮の新陳代謝を低下させたり、真皮細胞におけるコラーゲン分解を亢進するために、真皮のコラーゲンを低下させたりすることが考えられた。すなわち、ｓｔｒａｔｉｆｉｎは、紫外線曝露により皮膚損傷を引き起こす主要タンパクの一種であることが推察された。 The present inventor has clarified that exposure of ultraviolet light to epidermal cells increases the amount of stratifin mRNA expression and the amount of protein. It was also clarified that the expression of stratifin is enhanced in the face of the exposed area as compared with the inside of the upper arm of the unexposed area. From these results, the exposure of ultraviolet rays increases the amount of stratifin, which reduces the metabolism of the epidermis to stop the progression of the cell cycle in the epidermal cells, or lowers the collagen of the dermis to enhance the collagen degradation in the dermal cells. It was thought that it would be done. That is, it was speculated that stratifin is one of the major proteins that cause skin damage by UV exposure.
以上から、被験対象の皮膚からテープストリップ等により採取したｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子の発現産物であるｍＲＮＡ発現量を指標にして、紫外線曝露による皮膚損傷の程度を検査することが可能であると考えられた。 Based on the above, it is considered possible to examine the degree of skin damage due to UV exposure, using the expression level of mRNA, which is the expression product of the gene encoding the stratifin protein, collected from the skin of the test subject with a tape strip as an index. Was given.
本発明の「皮膚損傷」とは、外的及び内的要因により起こる皮膚組織の物理的な損傷を示す。例えば、切り傷、擦り傷、刺し傷、火傷、咬傷、挫傷、日焼け、色素沈着、シワ・タルミ、良性腫瘍、前がん症（日光角化症、悪性黒子）、皮膚がん等が挙げられる。 The “skin damage” of the present invention refers to physical damage to skin tissue caused by external and internal factors. For example, cuts, abrasions, punctures, burns, bites, bruising, sunburns, pigmentation, wrinkles/talumi, benign tumors, precancers (keratokeratosis, malignant lentis), skin cancer and the like can be mentioned.
又、本発明の「紫外線曝露による皮膚損傷」とは、皮膚損傷のうち、紫外線曝露が原因により生じる皮膚損傷を示す。例えば、日焼け、色素沈着、シワ・タルミ、良性腫瘍、前がん症（日光角化症、悪性黒子）、皮膚がん等が挙げられる。 Further, the term “skin damage due to exposure to ultraviolet light” of the present invention refers to, among skin damage, skin damage caused by exposure to ultraviolet light. For example, sunburn, pigmentation, wrinkle/tarumi, benign tumor, precancerous disease (actinic keratosis, malignant lentis), skin cancer, etc.
本発明における「遺伝子発現の定量化」とは、遺伝子の転写産物であるｍＲＮＡ発現量を測定することである。又は、遺伝子の発現産物であるタンパク量を測定しても良い。 The “quantification of gene expression” in the present invention is to measure the expression amount of mRNA which is a transcription product of a gene. Alternatively, the amount of protein that is the expression product of the gene may be measured.
ｍＲＮＡ発現量の測定には、マイクロアレイ解析や、逆転写反応を行った後にポリメラーゼ連鎖反応法（ＰＣＲ法）を行う方法等を用いることができる。これらの方法は、常法に従って実施すれば良い。各方法について様々なプロトコールが報告されており、当業者であれば公知のプロトコールに従い、又は、公知のプロトコールを適宜修正・変更したプロトコールにより、適切な測定系を構築し、実施することができる。尚、ｍＲＮＡ発現量の測定による遺伝子発現の定量化の詳細については後述する。 Microarray analysis, a method of performing a polymerase chain reaction method (PCR method) after performing a reverse transcription reaction, or the like can be used for measuring the mRNA expression level. These methods may be carried out according to conventional methods. Various protocols have been reported for each method, and those skilled in the art can construct and carry out an appropriate measurement system according to a known protocol or a protocol in which a known protocol is appropriately modified or changed. The details of quantification of gene expression by measuring the amount of mRNA expression will be described later.
一方、タンパク量の測定であれば、例えば、蛍光物質、色素、酵素等を利用する免疫染色法、ウエスタンブロット法、免疫測定法（例えば、ＥＬＩＳＡ法やＥＩＡ法等）等を用いることができる。これらの方法についても、常法に従って実施すれば良い。各方法について様々なプロトコールが報告されており、当業者であれば公知のプロトコールに従い、又は、公知のプロトコールを適宜修正・変更したプロトコールにより、適切な測定系を構築し、実施することができる。 On the other hand, if the amount of protein is to be measured, for example, an immunostaining method using a fluorescent substance, a dye, an enzyme, etc., a Western blotting method, an immunoassay method (eg, ELISA method, EIA method, etc.) can be used. These methods may be carried out according to the usual method. Various protocols have been reported for each method, and those skilled in the art can construct and carry out an appropriate measurement system according to a known protocol or a protocol in which a known protocol is appropriately modified or changed.
本発明に用いる「被験対象の皮膚」であるが、非露光部位の皮膚については、紫外線に曝されにくく、光老化の影響が比較的少ない部位の皮膚、例えば、上腕内側部、前腕内側部、背部、臀部又は大腿内側部等、日常的に衣服に覆われている部位の皮膚が望ましい。又、露光部位の皮膚については、紫外線に曝されやすく、光老化の影響が比較的多い部位の皮膚、例えば、顔面又は首筋等、日常的に衣服から露出している部位の皮膚が望ましい。非露光部位及び露光部位共に、このような試料（組織或いは細胞）の培養物であっても良い。 Although it is the "skin of the subject" used in the present invention, for the skin of the unexposed site, it is difficult to be exposed to ultraviolet rays, the skin of the site relatively less affected by photoaging, for example, the inner part of the upper arm, the inner part of the forearm, It is desirable that the skin is the skin that is routinely covered with clothes, such as the back, buttocks, or inner thigh. As for the skin of the exposed area, it is desirable that the skin is easily exposed to ultraviolet rays and has a relatively large effect of photoaging, for example, the skin of the face, the neck or the like, which is routinely exposed from clothes. Both the unexposed region and the exposed region may be a culture of such a sample (tissue or cell).
本発明に用いる被験対象の皮膚から採取した「角質」とは、皮膚の中で最外層に存在する角質層における細胞を示す。皮膚の中で最外層に存在する角質におけるｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子の発現を解析することから、本発明の皮膚損傷検査法は、皮膚をあまり傷つけず、且つ、容易に実施可能なものになる。 “Corneum” collected from the skin of the test subject used in the present invention refers to cells in the stratum corneum existing in the outermost layer in the skin. Since the expression of the gene encoding the stratifin protein in the outermost layer of the skin, which is the stratum corneum, is analyzed, the skin damage test method of the present invention does not damage the skin much and can be easily carried out. ..
皮膚からの角質の採取方法は特に限定されないが、可能な限り、非侵襲的な採取方法を採用することが好ましい。すなわち、角質の採取の際、顆粒層や有棘層等の角質層以外の部分を、可能な限り、傷つけないことが望まれる。非侵襲的な採取方法としては、例えば、粘着性のテープや接着剤等を皮膚へ付着させた後に剥がす方法や、粗面の材料（例えば、不織布等）にて皮膚表面を擦る方法により角質を採取することができる。非常に簡便に実施できる点において、前者の方法は特に好ましい。尚、以降に実施される遺伝子発現の定量化に影響しないものである限り、粘着性のテープや接着剤に用いられる材料（接着成分）は特に限定されない。 The method for collecting keratin from the skin is not particularly limited, but it is preferable to adopt a non-invasive method for collecting as much as possible. That is, when collecting keratin, it is desired that the part other than the horny layer such as the granular layer or the spinous layer is not damaged as much as possible. Non-invasive collection methods include, for example, a method of adhering adhesive tape or adhesive to the skin and then peeling it off, or a method of rubbing the skin surface with a rough surface material (for example, non-woven fabric) to remove keratin. Can be collected. The former method is particularly preferable because it can be carried out very easily. The material (adhesive component) used for the adhesive tape or adhesive is not particularly limited as long as it does not affect the quantification of gene expression performed thereafter.
本発明の「皮膚損傷検査法」は、好ましくは、以下の一連のステップ（１）〜（３）により実施される。
（１）被験対象の皮膚（非露光部位及び露光部位）から採取した角質を用意するステップ
（２）前記角質から抽出したｍＲＮＡを試料として遺伝子発現の定量化を行い、ｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子の発現量を算出するステップ
（３）ステップ（２）にて算出した発現量を用いて、紫外線曝露による皮膚損傷の程度を検査するステップ
The "skin damage inspection method" of the present invention is preferably carried out by the following series of steps (1) to (3).
(1) Step of preparing keratin collected from the skin (non-exposed site and exposed site) of a test subject (2) Quantification of gene expression using mRNA extracted from the keratin as a sample to determine the gene encoding stratifin protein Step (3) of calculating the expression level A step of inspecting the degree of skin damage due to UV exposure using the expression level calculated in the step (2)
ステップ（１）では、被験対象の皮膚から採取した角質を用意する。以下に、角質の採取法、及び、ＲＮＡの抽出法の具体例を示す。
（ａ）顆粒層や有棘層等を傷つけないように、最外層である角質層の一部を剥離する。例えば、粘着性のテープを皮膚へ付着させた後に剥がし（必要に応じて、数回繰り返す）、角質を採取する。
（ｂ）採取した角質を、ＳＬＳ、β−メルカプトエタノール、グアニジンイソチオシアネート等を含む溶解性緩衝液に浸した後、タンパク質分解酵素を加え、反応させる。
（ｃ）反応終了後、例えば、超音波破砕機等の物理的な力により、角質を破砕する。
（ｄ）角質破砕物を含む溶液から、周知の核酸抽出法に準じた方法や市販されたキットを用いた方法により、ＲＮＡを抽出する。例えば、グアニジンイソチオシアネート、フェノール又はクロロホルムを用いたＲＮＡ抽出法、ニッポンジーン社のＩＳＯＧＥＮ、Ｉｎｖｉｔｒｏｇｅｎ社のＴｒｉｚｏｌ Ｒｅａｇｅｎｔ、或いは、ＱＩＡＧＥＮ社のＲＮｅａｓｙ Ｋｉｔ、Ａｍｂｉｏｎ社のＲＮＡｑｕｅｏｕｓ−４ＰＣＲ Ｋｉｔ等を用いる方法によりＲＮＡを抽出する。
（ｅ）必要に応じて、ＤＮＡ分解酵素を反応させ、ＤＮＡを除去する。
（ｆ）必要に応じて、エタノール沈殿等の核酸濃縮法を用い、ＲＮＡを濃縮する。
In step (1), keratin collected from the skin of the subject is prepared. Specific examples of the method for collecting keratin and the method for extracting RNA are shown below.
(A) A part of the stratum corneum, which is the outermost layer, is peeled off so as not to damage the granular layer or the spinous layer. For example, the adhesive tape is attached to the skin and then peeled off (repeated several times as necessary), and the keratin is collected.
(B) The collected keratin is immersed in a solubility buffer containing SLS, β-mercaptoethanol, guanidine isothiocyanate, etc., and then a proteolytic enzyme is added and reacted.
(C) After the reaction is completed, the keratin is crushed by a physical force such as an ultrasonic crusher.
(D) RNA is extracted from a solution containing crushed keratin by a method according to a well-known nucleic acid extraction method or a method using a commercially available kit. For example, RNA is extracted by a method using RNA extraction method using guanidine isothiocyanate, phenol or chloroform, ISOGEN manufactured by Nippon Gene, Trizol Reagent manufactured by Invitrogen, or RNeasy Kit manufactured by QIAGEN, RNAqueous-4PCR Kit manufactured by Ambion. To do.
(E) If necessary, a DNA degrading enzyme is reacted to remove the DNA.
(F) If necessary, RNA is concentrated using a nucleic acid concentration method such as ethanol precipitation.
ステップ（２）では、採取した角質から抽出したｍＲＮＡを試料として、遺伝子発現の定量化を行う。そして、当該遺伝子発現の定量化により、特定の遺伝子の発現量を算出する。このように、本発明の一態様では、角質中に残存するｍＲＮＡ発現量を指標にした遺伝子発現の定量化を行う。尚、遺伝子発現の定量化では、「遺伝子の発現量」は絶対値又は相対値（比較対象又は標準の発現量との比率や差等）として算出される。 In step (2), gene expression is quantified using mRNA extracted from the collected keratin as a sample. Then, the expression level of the specific gene is calculated by quantifying the gene expression. Thus, in one aspect of the present invention, gene expression is quantified by using the amount of mRNA expression remaining in the corneum as an index. In the quantification of gene expression, the “gene expression level” is calculated as an absolute value or a relative value (a ratio or a difference with the comparison target or standard expression level).
ここで、角質から抽出したｍＲＮＡを用いた遺伝子発現の定量化方法の具体例を、以下に示す。 Here, a specific example of a method for quantifying gene expression using mRNA extracted from keratin will be shown below.
ＲＴ−ＰＣＲを用いた方法
（ａ）抽出したＲＮＡを鋳型に、逆転写酵素を用いてｃＤＮＡを合成する。
（ｂ）ｃＤＮＡを鋳型に、ターゲットとなる遺伝子に対応するプライマーを用いてＰＣＲ反応を行い、目的の遺伝子に対応するＤＮＡ断片を得る。
（ｃ）内部標準となるような遺伝子（β−アクチン、ＧＡＰＤＨ、ケラチン６Ｂ等）の反応も並行して行う。
（ｄ）得られたＤＮＡ断片の電気泳動を行った後、エチジウムブロマイド等にて染色してバンドの強度を測定し、その遺伝子の発現量とする。
（ｅ）必要に応じて、ＳＹＢＲ ｇｒｅｅｎ等の蛍光色素やＴａｑｍａｎ ｐｒｏｂｅ等の蛍光プローブを用いて定量ＰＣＲ反応を行い、発現量を定量する。
（ｆ）内部標準となる遺伝子の発現量に対する、目的の遺伝子の発現量の比率を算出する。
Method using RT-PCR (a) cDNA is synthesized using the extracted RNA as a template and reverse transcriptase.
(B) Using the cDNA as a template and a PCR reaction using a primer corresponding to the target gene, a DNA fragment corresponding to the target gene is obtained.
(C) Reactions of genes (β-actin, GAPDH, keratin 6B, etc.) that serve as internal standards are also performed in parallel.
(D) After the obtained DNA fragment is electrophoresed, it is stained with ethidium bromide and the intensity of the band is measured to obtain the expression level of the gene.
(E) If necessary, a quantitative PCR reaction is performed using a fluorescent dye such as SYBR green or a fluorescent probe such as Taqman probe to quantify the expression level.
(F) The ratio of the expression level of the target gene to the expression level of the gene serving as the internal standard is calculated.
ステップ（３）では、ステップ（２）にて算出した発現量を用いて、紫外線曝露による皮膚損傷の程度を検査する。すなわち、ステップ（２）にて算出した発現量を用いて、露光部位／非露光部位のｓｔｒａｔｉｆｉｎ ｍＲＮＡの発現比率を計算する。そして、その発現比率が、紫外線曝露による皮膚損傷の程度が関連付けられた複数の区分の中のいずれに該当するのかを調べる。区分設定に関する具体例を以下に示す。
（例）皮膚損傷の程度（低い）：比率＜ａ、（中程度）：ａ≦比率＜ｂ、（高い）：ｂ≦比率
In step (3), the expression level calculated in step (2) is used to inspect the degree of skin damage due to UV exposure. That is, using the expression level calculated in step (2), the expression ratio of the stratifin mRNA in the exposed site/non-exposed site is calculated. Then, it is examined which of the plurality of categories the expression ratio corresponds to, the degree of skin damage due to UV exposure. A specific example of setting categories is shown below.
(Example) Degree of skin damage (low): ratio <a, (moderate): a ≤ ratio <b, (high): b ≤ ratio
（例）では区分数を３としているが、区分数は特に限定されるものではない。例えば、区分数を２〜１０のいずれかにすることができる。区分数、及び、各区分に関連付けられる基準発現量の値の範囲は、予備実験の結果を基に任意に設定可能である。 Although the number of categories is 3 in the example, the number of categories is not particularly limited. For example, the number of sections can be set to any of 2 to 10. The number of categories and the range of values of the reference expression level associated with each category can be arbitrarily set based on the results of preliminary experiments.
以下、本発明を効果的に説明するために、実験例を挙げる。尚、本発明はこれにより限定されるものではない。 Hereinafter, in order to effectively explain the present invention, experimental examples will be given. The present invention is not limited to this.
実験例１ ヒトケラチノサイトにおける、紫外線曝露によるｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子の発現変化
培養細胞を用いて、紫外線曝露によるｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子発現の定量化を行った。すなわち、６ｃｍシャーレにヒトケラチノサイトを播種し、３７℃、５％ＣＯ２条件下にて培養した。コンフルエントな状態になったところで、３０ｍＪ／ｃｍ２のＵＶＢを曝露し、更に６時間培養した。その後、細胞よりＴｒｉｚｏｌ Ｒｅａｇｅｎｔ（Ｉｎｖｉｔｒｏｇｅｎ社）を用いて総ＲＮＡの抽出を行い、細胞から抽出した総ＲＮＡを基に、ＲＴ−ＰＣＲ法によりｓｔｒａｔｉｆｉｎ ｍＲＮＡ発現量の測定を行った。同時に、内部標準として、ＧＡＰＤＨ ｍＲＮＡ発現量の測定を行った。尚、各遺伝子の発現量の測定に使用したプライマーは次の通りである。
Experimental Example 1 Change in expression of gene encoding stratifin protein in human keratinocytes due to exposure to ultraviolet rays The expression of gene encoding stratifin protein due to exposure to ultraviolet rays was quantified using cultured cells. That is, human keratinocytes were seeded on a 6 cm dish and cultured under conditions of 37° C. and 5% CO 2 . When the cells became confluent, they were exposed to 30 mJ/cm 2 of UVB and further cultured for 6 hours. Then, total RNA was extracted from the cells using Trizol Reagent (Invitrogen), and based on the total RNA extracted from the cells, the amount of expression of stratifin mRNA was measured by the RT-PCR method. At the same time, the GAPDH mRNA expression level was measured as an internal standard. The primers used for measuring the expression level of each gene are as follows.
ｓｔｒａｔｉｆｉｎ用のプライマーセット
ＡＧＡＴＧＣＣＧＣＣＣＡＣＣＡＡ（配列番号１）
ＡＴＣＴＣＧＴＡＧＴＧＧＡＡＧＡＣＧＧＡＡＡＡＧ（配列番号２）
ＧＡＰＤＨ用のプライマーセット
ＴＧＡＡＣＧＧＧＡＡＧＣＴＣＡＣＴＧＧ（配列番号３）
ＴＣＣＡＣＣＡＣＣＣＴＧＴＴＧＣＴＧＴＡ（配列番号４）
Primer set for Stratifin AGATGCCGCCCACCAA (SEQ ID NO: 1)
ATCTCGTAGTGGAAGACGGAAAAG (SEQ ID NO: 2)
Primer set for GAPDH TGAACGGGAAGCTCACTGG (SEQ ID NO: 3)
TCCACCACCCTGTTGCTGTA (SEQ ID NO: 4)
その結果を表１に示す。図示の通り、ヒトケラチノサイトにＵＶＢを曝露すると、ｓｔｒａｔｉｆｉｎ ｍＲＮＡの発現量は増加した。 The results are shown in Table 1. As shown in the figure, exposure of UVB to human keratinocytes increased the expression level of stratifin mRNA.
実験例２ ヒトケラチノサイトにおける、紫外線曝露によるｓｔｒａｔｉｆｉｎタンパクの変化
培養細胞を用いて、紫外線曝露によるｓｔｒａｔｉｆｉｎタンパクの定量化を行った。すなわち、６ｃｍシャーレにヒトケラチノサイトを播種し、３７℃、５％ＣＯ２条件下にて培養した。コンフルエントな状態になったところで、３０ｍＪ／ｃｍ２のＵＶＢを曝露し、更に２４時間培養した。その後、細胞よりＩＳＯＧＥＮ（ニッポンジーン社）を用いて総タンパクの抽出を行った。ｓｔｒａｔｉｆｉｎタンパク量の測定は、細胞から抽出した総タンパクを基にしてウエスタンブロット法により行った。すなわち、２ｍｇ／ｍｌの総タンパクをＳＤＳ−ＰＡＧＥ後、トランスファーメンブレンであるＩｍｍｏｂｉｌｏｎ−Ｐ（Ｍｉｌｌｉｐｏｒｅ社）にブロッティングした。次に、ａｎｔｉ−ｓｔｒａｔｉｆｉｎ抗体（Ａｎｔｉ−１４−３−３σ、ｃｌｏｎｅ ＣＳ１１２−２Ａ８、Ｍｉｌｌｉｐｏｒｅ社）と室温にて１時間反応させた後、Ｐｅｒｏｘｉｄａｓｅ−ｃｏｎｊｕｇａｔｅｄ ＡｆｆｉｎｉＰｌｕｓ Ｆ（ａｂ’）２ Ｆｒａｇｍｅｎｔ Ｇｏａｔ Ａｎｔｉ−Ｍｏｕｓｅ ＩｇＧ （Ｈ＋Ｌ）（Ｊａｃｋｓｏｎ ＩｍｍｕｎｏＲｅｓｅａｒｃｈ ＬＡＢＯＲＡＴＯＲＩＥＳ社）と室温にて１時間反応させた。その後、ＥＣＬ Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ Ｄｅｔｅｃｔｉｏｎ Ｒｅａｇｅｎｔｓ（Ａｍｅｒｓｈａｍ社）と室温にて反応させ、発光パターンを撮影した。撮影後、３０ｋＤ付近に現れたバンドを定量化した。
Experimental Example 2 Change of stratifin protein in human keratinocytes due to exposure to ultraviolet light The amount of stratifin protein due to exposure to ultraviolet light was quantified using cultured cells. That is, human keratinocytes were seeded on a 6 cm dish and cultured under conditions of 37° C. and 5% CO 2 . When it became confluent, it was exposed to 30 mJ/cm 2 of UVB and further cultured for 24 hours. Then, total protein was extracted from the cells using ISOGEN (Nippon Gene). The amount of stratifin protein was measured by Western blotting based on the total protein extracted from the cells. That is, 2 mg/ml of total protein was subjected to SDS-PAGE and then blotted onto a transfer membrane Immobilon-P (Millipore). Next, after reacting with an anti-stratifin antibody (Anti-14-3-3σ, clone CS112-2A8, Millipore) at room temperature for 1 hour, Peroxidase-conjugated AffiniPlus F(ab′)2 Fragment Goat Anti-Mouse Anti-. It was reacted with IgG (H+L) (Jackson ImmunoResearch LABORATORIES) at room temperature for 1 hour. Then, it was made to react with ECL Western Blotting Detection Reagents (Amersham) at room temperature, and the luminescence pattern was photographed. After photographing, the band that appeared around 30 kD was quantified.
その結果を表２に示す。図示の通り、ヒトケラチノサイトにＵＶＢを曝露すると、ｓｔｒａｔｉｆｉｎタンパク量は増加した。 The results are shown in Table 2. As shown, exposure of human keratinocytes to UVB increased the amount of stratifin protein.
実験例３ 皮膚のｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子の発現量の測定、及び、紫外線に対する防御への意識アンケート
１．角質に含まれるＲＮＡの抽出
男性被験者２９名（２６〜５９歳）の非露光部位である上腕内側、及び、露光部位である顔面の皮膚に、半径１ｃｍの円状のテープ（３Ｍ、Ｂｌｅｎｄｅｒｍ社）を１枚貼付し、十分に接着していることを確認した後、テープを剥がし取り、角質を含むテープを得た。この操作を４回繰り返し、同一部位から４枚のテープストリップサンプルを得た。得られた角質に含まれるＲＮＡを、ＲＮＡ抽出キット（ＲＮＡｑｕｅｏｕｓ−４ＰＣＲ Ｋｉｔ、Ａｍｂｉｏｎ社）を用いて抽出した。すなわち、角質の付着したテープ４枚を、テープごとＬｙｓｉｓ／Ｂｉｎｄｉｎｇ Ｓｏｌｕｔｉｏｎに入れ、ｐｒｏｔｅｉｎａｓｅ Ｋ（Ｉｎｖｉｔｒｏｇｅｎ社）を添加し、５６℃にてインキュベートした。その後、超音波破砕機を用いて分散させた後、６４％エタノールを加えて攪拌してテープを取り除き、角質抽出液を得た。付属のＦｉｌｔｅｒ ＣａｒｔｒｉｄｇｅにＲＮＡを吸着させた後、溶出液にてＲＮＡを溶出した。得られた溶出液にＤＮａｓｅＩを加え、残存しているＤＮＡを完全に除去した後、エタノールを用いてＲＮＡを沈殿させてＲＮＡを濃縮した。得られたＲＮＡのペレットを必要量の水に溶解し、角質に含まれる残存ＲＮＡを得た。
Experimental Example 3 Measurement of expression level of gene encoding stratifin protein in skin, and questionnaire regarding awareness of protection against ultraviolet rays 1. Extraction of RNA contained in horny layer 29-year-old male subjects (26 to 59 years old), a circular tape (3M, Blenderm) with a radius of 1 cm on the inner skin of the upper arm which is the unexposed site and the skin of the face which is the exposed site Was adhered, and after confirming that it was sufficiently adhered, the tape was peeled off to obtain a tape containing keratin. This operation was repeated 4 times to obtain 4 tape strip samples from the same site. The RNA contained in the obtained keratin was extracted using an RNA extraction kit (RNAqueous-4PCR Kit, Ambion). That is, four tapes with horny cells were put together in a Lysis/Binding Solution, proteinase K (Invitrogen) was added, and incubated at 56°C. Then, after dispersing using an ultrasonic crusher, 64% ethanol was added and stirred to remove the tape, to obtain a keratin extract. After adsorbing RNA to the attached Filter Cartridge, RNA was eluted with an eluent. DNase I was added to the obtained eluate to completely remove the remaining DNA, and then RNA was precipitated by using ethanol to concentrate the RNA. The obtained RNA pellet was dissolved in a required amount of water to obtain residual RNA contained in keratin.
２．角質に含まれるＲＮＡのＰＣＲ解析
上腕内側及び顔面の皮膚の角質より、上記実験例３の１．の方法にて残存ＲＮＡを得た。各ＲＮＡをＲＴ−ＰＣＲキット（ＳｕｐｅｒＳｃｒｉｐｔ ＩＩＩ Ｐｌａｔｉｎｕｍ ＳＹＢＲ Ｇｒｅｅｎ Ｔｗｏ−ｓｔｅｐ ｑＲＴ−ＰＣＲ ｋｉｔ、Ｉｎｖｉｔｒｏｇｅｎ社）を用いたＲＴ−ＰＣＲ反応に供し、ｓｔｒａｔｉｆｉｎ ｍＲＮＡの発現量を定量した。ｓｔｒａｔｉｆｉｎ ｍＲＮＡの発現量は、β−アクチン ｍＲＮＡの発現量にて規格化（ノーマライズ）した。尚、ｓｔｒａｔｉｆｉｎ ｍＲＮＡ発現の測定に使用したプライマーは、上記実験例１と同様のものである。β−アクチン ｍＲＮＡのプライマーは、次の通りである。
2. PCR analysis of RNA contained in keratin From the keratin of the skin on the inside of the upper arm and the face, 1. Residual RNA was obtained by the method. Each RNA was subjected to an RT-PCR reaction using an RT-PCR kit (SuperScript III Platinum SYBR Green Two-step qRT-PCR kit, Invitrogen) to quantify the expression level of stratifin mRNA. The expression level of stratifin mRNA was normalized by the expression level of β-actin mRNA. The primers used for measuring the expression of stratifin mRNA are the same as those used in Experimental Example 1. The primers for β-actin mRNA are as follows.
β−アクチン用のプライマーセット
ＡＧＣＧＣＧＧＣＴＡＣＡＧＣＴＴＣＡ（配列番号５）
ＣＴＴＡＡＴＧＴＣＡＣＧＣＡＣＧＡＴＴＴＣＣ（配列番号６）
Primer set for β-actin AGCGCGGCTACAGCTTCA (SEQ ID NO: 5)
CTTAATGTCACGCACGATTTC (SEQ ID NO: 6)
定量化はΔΔＣｔ法にて行い、ｓｔｒａｔｉｆｉｎ ｍＲＮＡの発現比率を、基準となるｓｔｒａｔｉｆｉｎ ｍＲＮＡ発現量に対する上腕内側及び顔面のｓｔｒａｔｉｆｉｎ ｍＲＮＡ発現量の比率として算出した。基準となるｓｔｒａｔｉｆｉｎ ｍＲＮＡ発現量は、ＢｉｏＣｈａｉｎ Ｉｎｓｔｉｔｕｔｅ社から購入したＴｏｔａｌ ＲＮＡ（Ｃａｔａｌｏｇ Ｎｏ．Ｒ１２３４２１８−Ｐ）から、上記実験例３の２．の方法にてｓｔｒａｔｉｆｉｎ ｍＲＮＡ発現量を算出したものを用いた。
上腕内側のΔΔＣｔ＝（上腕内側のｓｔｒａｔｉｆｉｎ ｍＲＮＡのＣｔ値―上腕内側のβ−アクチン ｍＲＮＡのＣｔ値）―（基準となるｓｔｒａｔｉｆｉｎ ｍＲＮＡのＣｔ値―基準となるβ−アクチン ｍＲＮＡのＣｔ値）
顔面のΔΔＣｔ＝（顔面のｓｔｒａｔｉｆｉｎ ｍＲＮＡのＣｔ値―顔面のβ−アクチン ｍＲＮＡのＣｔ値）―（基準となるｓｔｒａｔｉｆｉｎ ｍＲＮＡのＣｔ値―基準となるβ−アクチン ｍＲＮＡのＣｔ値）
遺伝子発現比率＝２−ΔΔＣｔ
The quantification was carried out by the ΔΔCt method, and the expression ratio of the stratifin mRNA was calculated as the ratio of the expression amount of the stratifin mRNA in the upper arm and the face to the reference expression amount of the stratifin mRNA. The amount of the expression of stratifin mRNA serving as a reference was obtained from Total RNA (Catalog No. R1234218-P) purchased from BioChain Institute, as described in 2. of Experimental Example 3. The amount of expression of stratifin mRNA was calculated by the method described in 1.
ΔΔCt of the inside of the upper arm=(Ct value of the stratifin mRNA inside the upper arm-Ct value of the β-actin mRNA inside the upper arm)-(Ct value of the standard stratifin mRNA-Ct value of the reference β-actin mRNA)
ΔΔCt of face=(Ct value of face stratifin mRNA-Ct value of face β-actin mRNA)-(Ct value of reference stratifin mRNA-Ct value of reference β-actin mRNA)
Gene expression ratio=2- ΔΔCt
その結果を表３に示す。図示の通り、ｓｔｒａｔｉｆｉｎ ｍＲＮＡ及びβ−アクチン ｍＲＮＡ共に、上腕内側及び顔面から検出可能であった。すなわち、ｓｔｒａｔｉｆｉｎの遺伝子発現産物は、テープストリップ等によりヒトの皮膚から容易に、且つ、非侵襲的に採取することができることが分かった。又、非露光部位である上腕内側と比較して、露光部位である顔面はｓｔｒａｔｉｆｉｎ ｍＲＮＡ発現比率が高かった。すなわち、紫外線曝露により、ｓｔｒａｔｉｆｉｎの発現が亢進することが分かった。 The results are shown in Table 3. As shown in the figure, both the stratifin mRNA and the β-actin mRNA were detectable from the inside of the upper arm and the face. That is, it was found that the gene expression product of stratifin can be easily and non-invasively collected from human skin by a tape strip or the like. In addition, the ratio of expression of stratifin mRNA was higher in the face, which was the exposed area, than in the inner arm, which was the unexposed area. That is, it was found that the expression of stratifin was enhanced by exposure to ultraviolet light.
３．紫外線に対する防御への意識アンケート
上記実験例３の１．及び２．と同じ男性被験者のうち、顔面／上腕内側のｓｔｒａｔｉｆｉｎ ｍＲＮＡの発現比率が高い８名及び低い８名に、紫外線に対する防御への意識アンケートを行った。すなわち、「屋外で行動する時、日焼け止め化粧品を塗るか？」の質問に対しては、「必ず塗る」、「時々塗る」、「ほとんど塗らない」の３つの解答を選択させた。又、「日焼け止め化粧品をこまめに塗り直すか？」の質問に対しては、「こまめに塗り直す」、「ほとんど塗り直さない」の２つの解答を選択させた。
3. Questionnaire on awareness of protection against ultraviolet rays 1. And 2. Among the same male subjects, a consciousness questionnaire on protection against ultraviolet rays was conducted on 8 persons with high expression ratio of facial/inner brachial stratifin mRNA and 8 persons with low expression ratio. That is, in response to the question "Do you apply sunscreen cosmetics when you act outdoors?", three answers, "Always apply", "Apply occasionally", and "Almost do not apply" were selected. In addition, in response to the question, "Do you often repaint sunscreen cosmetics?", I asked them to choose two answers: "Repaint frequently" and "Repaint little."
その結果を表４及び５に示す。「屋外で行動する時、日焼け止め化粧品を塗るか？」の質問に対しての解答である「必ず塗る」は○、「時々塗る」は△、「ほとんど塗らない」は×で示した。又、「日焼け止め化粧品をこまめに塗り直すか？」の質問に対しての解答である「こまめに塗り直す」は○、「ほとんど塗り直さない」は×で示した。 The results are shown in Tables 4 and 5. The answer to the question "Do you apply sunscreen cosmetics when you act outdoors?" is "○", "sometimes" is "△", and "almost never" is "x". In addition, the answer to the question "Do you frequently repaint sunscreen cosmetics?" is "○" and "X almost never" is "X".
表４及び５から、顔面／上腕内側のｓｔｒａｔｉｆｉｎ ｍＲＮＡの発現比率が高い群は、紫外線に対する防御への意識が低いと推測された。言い換えると、比較的、紫外線に曝露される環境下にあるため、顔面／上腕内側のｓｔｒａｔｉｆｉｎ ｍＲＮＡの発現比率が高く、皮膚損傷が大きいと考えられた。一方、顔面／上腕内側のｓｔｒａｔｉｆｉｎ ｍＲＮＡの発現比率が低い群は、紫外線に対する防御への意識が高いと推測された。言い換えると、比較的、紫外線に曝露される環境下にないため、顔面／上腕内側のｓｔｒａｔｉｆｉｎ ｍＲＮＡの発現比率が低く、皮膚損傷が小さいと考えられた。 From Tables 4 and 5, it was inferred that the group in which the expression ratio of the stratifin mRNA on the face/inside the upper arm was high had low awareness of protection against ultraviolet rays. In other words, it was considered that the expression ratio of the stratifin mRNA on the face/inside the upper arm was high and the skin damage was large, because it was relatively exposed to the environment of ultraviolet rays. On the other hand, it was presumed that the group in which the expression ratio of the stratifin mRNA on the face/inside the upper arm was low had a high awareness of protection against ultraviolet rays. In other words, it was considered that the ratio of expression of stratifin mRNA on the face/inside the upper arm was low and the skin damage was small because it was not under the environment of being exposed to ultraviolet rays.
本発明の皮膚損傷検査法によれば、皮膚のｓｔｒａｔｉｆｉｎ ｍＲＮＡの発現量を指標にして、紫外線曝露による皮膚損傷の程度を検査することができる。この手法では、試料を得る際の痛み等を伴わず、非侵襲的に皮膚のサンプルを得ることができる。又、皮膚損傷の程度を検査することで、化粧品の使用方法を提示することが可能になり、皮膚損傷を予防・改善することができる。 According to the skin damage test method of the present invention, the degree of skin damage due to exposure to ultraviolet rays can be tested by using the expression level of skin stratifin mRNA as an index. With this method, a skin sample can be obtained non-invasively without causing pain when obtaining a sample. In addition, by examining the degree of skin damage, it becomes possible to present a method of using cosmetics, and it is possible to prevent or improve skin damage.
Claims (5)
（１）被験対象から皮膚のサンプルを得る工程、
（２）該サンプルのｓｔｒａｔｉｆｉｎタンパクをコードする遺伝子の発現を定量化する工程、
（３）工程（２）にて測定した遺伝子発現量を用いて、紫外線曝露による皮膚損傷の程度を検査する工程
を含む方法。 A method for examining the degree of skin damage due to ultraviolet exposure, based on the expression level of a gene encoding a stratifin protein in the skin, comprising:
(1) obtaining a skin sample from a test subject,
(2) quantifying the expression of a gene encoding the stratifin protein in the sample,
(3) A method including a step of inspecting the degree of skin damage due to UV exposure using the gene expression level measured in step (2).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010185680A JP5653684B2 (en) | 2010-08-21 | 2010-08-21 | How to check the degree of skin damage |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010185680A JP5653684B2 (en) | 2010-08-21 | 2010-08-21 | How to check the degree of skin damage |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2012039970A true JP2012039970A (en) | 2012-03-01 |
| JP5653684B2 JP5653684B2 (en) | 2015-01-14 |
Family
ID=45896998
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2010185680A Active JP5653684B2 (en) | 2010-08-21 | 2010-08-21 | How to check the degree of skin damage |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5653684B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021210651A1 (en) | 2020-04-17 | 2021-10-21 | 国立医薬品食品衛生研究所長が代表する日本国 | Protein diagnostic biomarker for severe drug eruption |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002054078A2 (en) * | 2001-01-03 | 2002-07-11 | Henkel Kommandigesellschaft Auf Aktien | Method for determining skin ageing in vitro |
| JP2008178390A (en) * | 2006-12-27 | 2008-08-07 | Nippon Menaade Keshohin Kk | Method of evaluating skin condition and application of the same |
-
2010
- 2010-08-21 JP JP2010185680A patent/JP5653684B2/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002054078A2 (en) * | 2001-01-03 | 2002-07-11 | Henkel Kommandigesellschaft Auf Aktien | Method for determining skin ageing in vitro |
| JP2008178390A (en) * | 2006-12-27 | 2008-08-07 | Nippon Menaade Keshohin Kk | Method of evaluating skin condition and application of the same |
Non-Patent Citations (2)
| Title |
|---|
| JPN6014038110; Ｏｎｃｏｇｅｎｅ ｖｏｌ．２２， ｎｏ．１９, 2003, ｐｐ．２９９３-３００６ * |
| JPN6014038111; ＰＬｏＳ ＯＮＥ ｖｏｌ．５， ｎｏ．５, 2010, ｐｐ．ｅ１０７７６（１-１０） * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021210651A1 (en) | 2020-04-17 | 2021-10-21 | 国立医薬品食品衛生研究所長が代表する日本国 | Protein diagnostic biomarker for severe drug eruption |
Also Published As
| Publication number | Publication date |
|---|---|
| JP5653684B2 (en) | 2015-01-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| McLean et al. | “Sunburn” freckles, café-au-lait macules, and other pigmented lesions of schoolchildren: The Vancouver Mole Study | |
| Boniol et al. | Variation in incidence and fatality of melanoma by season of diagnosis in New South Wales, Australia | |
| JP5027528B2 (en) | Test method for skin aging | |
| WO2017165199A1 (en) | Next-generation biomarkers to detect sun damage and predict skin cancer risk | |
| US11892447B2 (en) | Method for evaluating the effects of dehydration on children's skin | |
| JP2020531027A (en) | How to diagnose skin that shows signs of aging | |
| Xin et al. | Correlation analysis between advanced glycation end products detected noninvasively and skin aging factors | |
| JP5653783B2 (en) | Evaluation method of water retention ability in skin epidermis | |
| JP6462437B2 (en) | Evaluation method of skin dryness | |
| JP5653684B2 (en) | How to check the degree of skin damage | |
| EP2893345B1 (en) | Method of evaluating cellulite and method of evaluating cellulite-effective drug using fibulin-3 and/or sarcoglycan gamma as an indicator | |
| JP5775538B2 (en) | Evaluation method of resistance to redness (redness) generation and reduction of whiteness induced by UV exposure | |
| Grover et al. | The nail as an investigative tool in medicine: What a dermatologist ought to know | |
| TWI600904B (en) | Method of evaluating degree of skin stress accumulation | |
| JP5693821B2 (en) | Method for measuring estrogen responsiveness of skin, and skin test method for predicting progress of skin aging such as formation of wrinkles / tarmi or rough texture | |
| CN109839507A (en) | A kind of biomarker detecting ultraviolet light injury and its application | |
| JP4473926B1 (en) | Evaluation method of skin darkening tendency by ultraviolet rays | |
| JP4446010B1 (en) | Evaluation method of resistance to skin sunburn by ultraviolet rays. | |
| JP5535571B2 (en) | Method for evaluating skin turnover and its use | |
| JP2010115178A (en) | Method of forecasting skin inflammation and application of the same | |
| Fan et al. | Evaluation and detection of early nail damage caused by nail enamel | |
| Yahya et al. | Profile of Non-Melanoma Skin Cancer and Malignant Melanoma at Dr. Mohammad Hoesin General Hospital Palembang from 2017-2019: A Retrospective Study | |
| Polat et al. | Prevalence of benign, precancerous and malignant skin tumors in the elderly population in Mugla | |
| JP2017216941A (en) | Diagnosis method of skin conditions | |
| Paterson et al. | Contemporary Approaches to Malignant Melanoma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20130619 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20140909 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20141014 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20141118 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20141119 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 5653684 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |