JP5658696B2 - Evaluation method of skin stress accumulation - Google Patents
Evaluation method of skin stress accumulation Download PDFInfo
- Publication number
- JP5658696B2 JP5658696B2 JP2012012033A JP2012012033A JP5658696B2 JP 5658696 B2 JP5658696 B2 JP 5658696B2 JP 2012012033 A JP2012012033 A JP 2012012033A JP 2012012033 A JP2012012033 A JP 2012012033A JP 5658696 B2 JP5658696 B2 JP 5658696B2
- Authority
- JP
- Japan
- Prior art keywords
- skin
- hsp27
- stratum corneum
- expression level
- stress
- 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.)
- Active
Links
- 238000009825 accumulation Methods 0.000 title claims description 40
- 238000011156 evaluation Methods 0.000 title claims description 25
- 210000003491 skin Anatomy 0.000 claims description 110
- 108010045100 HSP27 Heat-Shock Proteins Proteins 0.000 claims description 102
- 210000000434 stratum corneum Anatomy 0.000 claims description 86
- 230000014509 gene expression Effects 0.000 claims description 53
- 238000000034 method Methods 0.000 claims description 40
- 230000037307 sensitive skin Effects 0.000 claims description 26
- 238000005259 measurement Methods 0.000 claims description 13
- 238000005070 sampling Methods 0.000 claims description 10
- 102000002812 Heat-Shock Proteins Human genes 0.000 claims description 7
- 108010004889 Heat-Shock Proteins Proteins 0.000 claims description 7
- 230000000638 stimulation Effects 0.000 claims description 5
- 102100039165 Heat shock protein beta-1 Human genes 0.000 claims 2
- 102000005623 HSP27 Heat-Shock Proteins Human genes 0.000 description 100
- 230000035882 stress Effects 0.000 description 57
- 108090000623 proteins and genes Proteins 0.000 description 41
- 102000004169 proteins and genes Human genes 0.000 description 41
- 238000002474 experimental method Methods 0.000 description 22
- 239000002537 cosmetic Substances 0.000 description 18
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 15
- 239000000284 extract Substances 0.000 description 15
- 230000036074 healthy skin Effects 0.000 description 15
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 15
- 229940083575 sodium dodecyl sulfate Drugs 0.000 description 14
- 238000011002 quantification Methods 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 238000000265 homogenisation Methods 0.000 description 9
- 206010012438 Dermatitis atopic Diseases 0.000 description 8
- 201000008937 atopic dermatitis Diseases 0.000 description 8
- 230000005068 transpiration Effects 0.000 description 8
- 230000035945 sensitivity Effects 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 230000000903 blocking effect Effects 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000000751 protein extraction Methods 0.000 description 6
- 206010015150 Erythema Diseases 0.000 description 5
- 235000010724 Wisteria floribunda Nutrition 0.000 description 5
- 210000000245 forearm Anatomy 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 4
- 230000037311 normal skin Effects 0.000 description 4
- 238000002731 protein assay Methods 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 241000283707 Capra Species 0.000 description 3
- 201000004624 Dermatitis Diseases 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 239000002033 PVDF binder Substances 0.000 description 3
- 208000003251 Pruritus Diseases 0.000 description 3
- 206010040880 Skin irritation Diseases 0.000 description 3
- 239000000853 adhesive Substances 0.000 description 3
- 230000001070 adhesive effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000011544 gradient gel Substances 0.000 description 3
- 230000007794 irritation Effects 0.000 description 3
- 230000007803 itching Effects 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- BBKDWPHJZANJGB-IKJXHCRLSA-N quizalofop-P-tefuryl Chemical compound O=C([C@H](OC=1C=CC(OC=2N=C3C=CC(Cl)=CC3=NC=2)=CC=1)C)OCC1CCCO1 BBKDWPHJZANJGB-IKJXHCRLSA-N 0.000 description 3
- 231100000475 skin irritation Toxicity 0.000 description 3
- 230000036556 skin irritation Effects 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 206010003645 Atopy Diseases 0.000 description 2
- 101100339887 Drosophila melanogaster Hsp27 gene Proteins 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 101150096895 HSPB1 gene Proteins 0.000 description 2
- 101001036709 Homo sapiens Heat shock protein beta-1 Proteins 0.000 description 2
- 102000011782 Keratins Human genes 0.000 description 2
- 108010076876 Keratins Proteins 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 230000003796 beauty Effects 0.000 description 2
- 210000001217 buttock Anatomy 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 231100000321 erythema Toxicity 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 102000053548 human HSPB1 Human genes 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 206010040882 skin lesion Diseases 0.000 description 2
- 231100000444 skin lesion Toxicity 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 238000000035 BCA protein assay Methods 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 230000007035 DNA breakage Effects 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000010201 Exanthema Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
- 102000005431 Molecular Chaperones Human genes 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000009223 counseling Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 201000005884 exanthem Diseases 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000006355 external stress Effects 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000010413 gardening Methods 0.000 description 1
- 230000008799 immune stress Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 230000019612 pigmentation Effects 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 206010037844 rash Diseases 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 230000005808 skin problem Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 230000035900 sweating Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 229910052724 xenon Inorganic materials 0.000 description 1
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/44—Detecting, measuring or recording for evaluating the integumentary system, e.g. skin, hair or nails
- A61B5/441—Skin evaluation, e.g. for skin disorder diagnosis
- A61B5/443—Evaluating skin constituents, e.g. elastin, melanin, water
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/20—Dermatological disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/40—Disorders due to exposure to physical agents, e.g. heat disorders, motion sickness, radiation injuries, altitude sickness, decompression illness
Description
本発明は、ヒト皮膚のストレス蓄積度の評価方法並びに、敏感肌の評価方法に関する。 The present invention relates to a method for evaluating the degree of stress accumulation in human skin and a method for evaluating sensitive skin.
ヒトの皮膚は常に外界と接触しており、さまざまな刺激を受けている。その刺激の代表的なものは紫外線である。紫外線は、外見上は大きな変化を肌に与えていない場合であっても、皮膚細胞のDNAの切断やコラーゲンの変性などを誘発し、皮膚老化の原因となっている。紫外線は常に肌にストレスを与え続けている。また女性にとっては、洗顔や化粧といった日常的な行為も皮膚に刺激を与えており、これらの刺激が肌には潜在的なストレスとして蓄積される。そしてこれらの刺激（ストレス）に対する抵抗性がなんらかの原因で減少すると敏感肌として認識されることとなる。このような皮膚の外的刺激の蓄積度を本発明では皮膚のストレス蓄積度という。一方敏感肌とは、明らかな皮膚病変はないが不利、有害な反応が起こりやすい肌として捉えられている。そしてこのような敏感肌の原因としてストレスの蓄積が一定レベルに到達すると、肌の感受性が高まるとも言われている。また敏感肌は、健常肌より外的刺激に対する抵抗性が低く、容易に皮膚トラブルを生ずる肌とも言える。近年、敏感肌を意識する女性が増加する傾向にあり、化粧品等についてもより刺激の少ないものが要望されるようになってきている。 Human skin is always in contact with the outside world and is subject to various stimuli. A typical example of the stimulus is ultraviolet rays. Ultraviolet rays cause skin aging by inducing DNA breakage and collagen degeneration of skin cells even when the skin is not significantly changed. Ultraviolet rays are constantly stressing the skin. For women, daily acts such as face washing and makeup also stimulate the skin, and these stimuli accumulate as potential stress on the skin. And if resistance to these stimuli (stress) decreases for some reason, it will be recognized as sensitive skin. In the present invention, the accumulation degree of such external stimulation of the skin is referred to as skin stress accumulation degree. Sensitive skin, on the other hand, is perceived as skin that has no obvious skin lesions but is prone to adverse and harmful reactions. It is also said that when the accumulation of stress reaches a certain level as a cause of such sensitive skin, the sensitivity of the skin increases. Sensitive skin is less resistant to external stimuli than healthy skin, and can be said to be skin that easily causes skin troubles. In recent years, there has been a tendency to increase the number of women who are conscious of sensitive skin, and there is a growing demand for cosmetics that are less irritating.
化粧品の販売現場では美容専門家による問診を行い、肌ストレスの蓄積状況や敏感肌か否かを評価して肌に与えるストレス（刺激）の少ない化粧品を選択してきた。しかし近年通信販売など、化粧品専門家の不在な販売方法が普及し、このため、肌のストレス状態を見極め、また敏感肌のレベルを専門家でなくとも簡便に評価することが望まれてきた。これが可能となれば、美容専門家の問診によらずとも、ユーザーの肌のストレス状態や敏感肌であるかどうかを評価して、適切な化粧品を自ら選択することが可能となる。またユーザーの肌により適した化粧品やケミカルピーリング剤を簡単に選択することができ、それによって、皮膚の炎症等の肌トラブルや副作用を回避し、化粧品によるスキンケアやケミカルピーリングの施術等の有益性をより高めることができる。このため、問診によらずとも、皮膚の外的刺激暴露履歴（肌ストレスの蓄積度）を評価し、さらに敏感肌であるかどうかを客観的に判別する方法が求められている。 At cosmetics sales sites, we have been interviewed by beauty specialists to evaluate the accumulation of skin stress and whether it is sensitive skin, and have selected cosmetics with less stress (stimulation) on the skin. However, in recent years, sales methods such as mail-order sales, in which cosmetic specialists are absent, have become widespread. For this reason, it has been desired to determine the stress state of the skin and to easily evaluate the level of sensitive skin without being an expert. If this is possible, it will be possible to evaluate the user's skin stress state and whether the skin is sensitive or not and to select appropriate cosmetics by themselves without consulting a beauty specialist. In addition, it is possible to easily select cosmetics and chemical peeling agents that are more suitable for the user's skin, thereby avoiding skin troubles and side effects such as skin irritation, and benefits such as cosmetic skin care and chemical peeling treatments. Can be increased. For this reason, there is a need for a method for evaluating the external irritation exposure history of skin (accumulation degree of skin stress) and objectively discriminating whether or not the skin is sensitive, regardless of an inquiry.
特許文献１には、皮膚に化粧料を塗布して、その後剥離してくる皮膚角層を回収して観察し、皮膚の敏感性を評価する方法が開示されている。しかしこの方法は、化粧品ごとにすべて検査しなければならず、またユーザーの肌ストレスの蓄積度は評価できない。 Patent Document 1 discloses a method for evaluating skin sensitivity by applying cosmetics to the skin, collecting and observing the exfoliated skin stratum corneum. However, this method must be inspected for each cosmetic product, and the accumulation degree of the user's skin stress cannot be evaluated.
特許文献２には、カプシノイドのような刺激性物質を皮膚に塗布して、敏感肌であるかどうかを評価するための方法が開示されている。しかしカプシノイドのような刺激性の強いものを用いる試験方法は、被験者にとっても好ましいものではない。 Patent Document 2 discloses a method for applying an irritating substance such as capsinoid to the skin and evaluating whether it is sensitive skin. However, a test method using a strongly irritating substance such as capsinoid is not preferable for the subject.
特許文献３には、顔の角層のカルプロテクチン存在量を測定して、敏感肌か否か評価する方法が開示されている。 Patent Document 3, by measuring the Ca pulp Rotekuchin abundance of the stratum corneum of the face, a method of assessing whether sensitive skin is disclosed.
非特許文献１、２は、ヒートショックプロテイン（HSP）27が界面活性剤であるソジウムラウリルサルフェート（SDS）による皮膚刺激に反応して皮膚で増加することが記載されている。 Non-Patent Documents 1 and 2 describe that heat shock protein (HSP) 27 increases in the skin in response to skin irritation by sodium lauryl sulfate (SDS), which is a surfactant.
上述したように、肌ストレスの蓄積度を評価し、敏感肌であるかどうかを客観的に評価する方法が望まれている。その評価方法は、外科的に皮膚を摘出する、皮膚に刺激を与える等のユーザーに負担を与える方法ではないことが望ましい。本発明は、ユーザーに負担を与えることなく、肌のストレスの蓄積度を評価し、さらに敏感肌かどうかを簡便かつ確実に評価することができる生化学的指標を用いた新規な評価方法を提供することを課題とする。 As described above, a method for evaluating the accumulation degree of skin stress and objectively evaluating whether or not the skin is sensitive is desired. It is desirable that the evaluation method is not a method that imposes a burden on the user, such as surgically removing the skin or irritating the skin. The present invention provides a novel evaluation method using a biochemical index that can evaluate the degree of accumulation of skin stress without burdening the user, and can easily and reliably evaluate whether the skin is sensitive or not. The task is to do.
本発明は以下の構成である。
（１）無作為に選抜した女性の頬から、非侵襲的に採取した皮膚角層のヒートショックプロテイン（HSP）27の発現量を測定し、平均値を超える値を示す場合、皮膚が紫外線刺激をストレスとして蓄積していると評価することを特徴とする、女性のヒト皮膚のストレス蓄積度の評価方法。
（２）無作為に選抜した女性の頬から、非侵襲的に採取した皮膚角層のHSP27の発現量を測定し、平均値を超える値を示す場合、敏感肌であるとの自己申告の裏づけとすることを特徴とする女性の敏感肌の評価方法。
（３）非侵襲的に皮膚角層を採取する採取工程と、前記採取工程で採取された角層細胞におけるHSP27の発現量を測定する測定工程と、あらかじめ設定した基準値と比較する比較工程と、を備えたことを特徴とする（１）又は（２）に記載の評価方法。
（４）皮膚の角層を採取する工程がテープストリッピング法によるものである（１）〜（３）のいずれかに記載の評価方法。
The present invention has the following configuration.
(1) When the expression level of heat shock protein (HSP) 27 in the stratum corneum collected non-invasively from a randomly selected female cheek is measured and shows a value exceeding the average value, the skin is stimulated with ultraviolet rays. A method for evaluating the degree of stress accumulation in human skin of a woman, characterized by evaluating the accumulation of stress as stress.
(2) Measure the expression level of HSP27 in the stratum corneum collected non-invasively from randomly selected women's cheeks, and if the value exceeds the average value, support the self-report that the skin is sensitive A method for evaluating sensitive skin of a woman, characterized by:
(3) Non-invasively collecting the stratum corneum , a measuring step for measuring the expression level of HSP27 in the stratum corneum cells collected in the collecting step, and a comparing step for comparing with a preset reference value The evaluation method according to (1) or (2) , wherein
(4) The evaluation method according to any one of (1) to (3) , wherein the step of collecting the stratum corneum of the skin is based on a tape stripping method.
本発明の評価方法によれば、肌ストレスの蓄積度を角層におけるHSP27の発現量を測定することで評価できる。またこのHSP27存在量を指標として、簡便に肌が敏感肌であるかどうかを評価できる。さらに、本発明の評価方法は、テープストリッピング法など簡便な操作方法で評価試料を採取するため、被験者の負担が少なく、だれでも簡単に評価することが可能となる。また生化学的な試験方法であり、誰が測定しても同一の結果が得られるため、従来のような専門家の面談方式によるカウンセリングが必要なくなる。 According to the evaluation method of the present invention, the accumulation degree of skin stress can be evaluated by measuring the expression level of HSP27 in the stratum corneum. Further, it is possible to easily evaluate whether the skin is sensitive skin by using the abundance of HSP27 as an index. Further, since the evaluation method of the present invention collects an evaluation sample by a simple operation method such as a tape stripping method, the burden on the subject is small, and anyone can easily evaluate. In addition, this is a biochemical test method, and the same result can be obtained regardless of who makes the measurement, so there is no need for conventional counseling by an expert interview method.
以下、本発明について詳細に説明する。
本発明の評価方法は、皮膚角層におけるHSP27の存在量を指標とすることを特徴としている。
HSP27とは一連のヒートショック蛋白質ファミリーの一つとして知られている分子量27KDの蛋白質で分子シャペロンとしての機能を有している。免疫ストレスにともなって皮膚での遺伝子発現が増加することが知られている。
Hereinafter, the present invention will be described in detail.
The evaluation method of the present invention is characterized by using the abundance of HSP27 in the stratum corneum as an index.
HSP27 is a protein with a molecular weight of 27 KD, which is known as one of a series of heat shock protein families, and has a function as a molecular chaperone. It is known that gene expression in the skin increases with immune stress.
本発明における敏感肌とは、明らかな皮膚病変はないが不利、有害な反応が起こりやすい肌をいう。また、健常肌より外界刺激に対する抵抗性が低く、容易に皮膚トラブルを生ずる肌とも言える。一方、健常肌とは、上記のような敏感肌の性質を示さない健康で正常な肌である。敏感肌においては、経皮水分蒸散量（TEWL）が高くなる、高周波伝導度（角層水分量）が低くなる等の傾向があることが知られている。また、敏感肌では、かぶれや肌荒れが起こる場合がある。 Sensitive skin in the present invention refers to skin that has no obvious skin lesions but is prone to adverse and harmful reactions. Moreover, it can be said that the skin is less susceptible to external stimuli than healthy skin and easily causes skin troubles. On the other hand, healthy skin is healthy and normal skin that does not exhibit the properties of sensitive skin as described above. It is known that in sensitive skin, there is a tendency that the transdermal moisture transpiration (TEWL) increases and the high-frequency conductivity (horny layer water content) decreases. In sensitive skin, rash and rough skin may occur.
角層は、皮膚の一番上にある組織であり、体の外からの異物や刺激から皮膚を守る働きを有している。 The stratum corneum is the tissue on the top of the skin and has a function of protecting the skin from foreign substances and irritation from outside the body.
本発明における評価対象部位は、角層が入手できる部分であれば、いかなる部位をも包含しうるが、主な部位としては顔面、頚部、上腕部を挙げることができる。従来の方法に従い、これらの部位の皮膚由来の角層を得ることができる。しかし、前述のように、外科的に皮膚を摘出する等の方法は、ユーザーに負担を与えるため、テープストリッピング、擦過等の簡便に角層を得られる方法が好ましい。 The evaluation target site in the present invention can include any site as long as the stratum corneum is available, but examples of the main site include the face, neck, and upper arm. According to the conventional method, the stratum corneum derived from the skin at these sites can be obtained. However, as described above, the method of surgically removing the skin places a burden on the user, and therefore, a method that can easily obtain the stratum corneum, such as tape stripping and rubbing, is preferable.
こうして用意した各試料におけるHSP27の発現量は、従来から知られている方法で測定することができる。例えば、HSP27に対する抗体との反応に基づくエンザイムイムノアッセイ、ラジオイムノアッセイ、ウエスタンブロッティング等の方法を用いることができる。 The expression level of HSP27 in each sample thus prepared can be measured by a conventionally known method. For example, methods such as enzyme immunoassay, radioimmunoassay, and Western blotting based on the reaction with an antibody against HSP27 can be used.
各試料からHSP27をそれ自体既知の生化学的方法、たとえば凍結融解法、超音波破砕法、ホモジュネート法等を介して可溶性画分を調製する。抽出後、速やかに測定する。 From each sample, the soluble fraction of HSP27 is prepared through biochemical methods known per se, such as freeze-thaw method, ultrasonic disruption method, homogenate method and the like. Measure immediately after extraction.
ストレスが蓄積された肌や敏感肌は、健常肌にくらべて角層におけるHSP27の発現量が有意に高いため、角層におけるHSP27の発現量の多寡を指標として、簡便にストレスの蓄積度又は肌が敏感肌であるかどうかを評価できる。本発明に用いる角層は、テープストリッピングといった角層の表層部分のみを角質テープで採取する簡単な方法で採取することができる。また、本発明は、皮膚に紫外線や化学物質等の刺激を与えることなくHSP27の発現量を測定できる。このため、ユーザーに負担を与えることなく、ユーザーの肌が敏感肌であるかどうかを評価することができる。特に、ユーザーの肌により適した化粧品やケミカルピーリング剤を簡単に選択することができ、それによって、皮膚の炎症等の肌トラブルや副作用を回避し、化粧品によるスキンケアや有益性をより発揮させることができる。 Since stress-accumulated skin and sensitive skin have significantly higher expression levels of HSP27 in the stratum corneum than healthy skin, the degree of stress accumulation or skin Can evaluate whether the skin is sensitive. The stratum corneum used in the present invention can be collected by a simple method such as tape stripping in which only the surface layer portion of the stratum corneum is collected with a keratin tape. In addition, the present invention can measure the expression level of HSP27 without stimulating the skin with ultraviolet rays or chemical substances. For this reason, it is possible to evaluate whether the user's skin is sensitive skin without imposing a burden on the user. In particular, it is possible to easily select cosmetics and chemical peeling agents that are more suitable for the user's skin, thereby avoiding skin troubles and side effects such as skin irritation and making skincare and benefits of cosmetics more effective. it can.
本発明の評価方法は、角層を採取する採取工程と、前記採取工程で採取された角層におけるHSP27の発現量を測定する測定工程と、前記測定工程で測定されたHSP27の発現量を健常肌の角層におけるHSP27の発現量と比較する比較工程から構成される。または、本発明の評価方法は、角層を採取する採取工程と、前記採取工程で採取された角層におけるHSP27の発現量を測定する測定工程と、前記測定工程で測定されたHSP27の発現量を標本集団の角層におけるHSP27の発現量の分布と比較する比較工程から構成される。以下にこの評価方法について説明する。 The evaluation method of the present invention includes a sampling step of sampling the stratum corneum, a measurement step of measuring the expression level of HSP27 in the stratum corneum sampled in the sampling step, and a healthy expression level of HSP27 measured in the measurement step. Comparing with the expression level of HSP27 in the stratum corneum of the skin. Alternatively, the evaluation method of the present invention includes a sampling step of sampling the stratum corneum, a measurement step of measuring the expression level of HSP27 in the stratum corneum sampled in the sampling step, and the expression level of HSP27 measured in the measurement step Is compared with the distribution of the expression level of HSP27 in the stratum corneum of the sample population. This evaluation method will be described below.
まず、上記のようにして測定されたHSP27の発現量を健常肌の角層におけるHSP27の発現量と比較する。また、被験者のある評価対象部位の角層におけるHSP27の発現量を測定し、測定されたHSP27の発現量を、健常肌を有する人の同一評価対象部位の角層におけるHSP27の発現量と比較してもよい。健常肌を有する人の角層のHSP27の発現量は、複数人の健常肌を有する人からのデータの平均値を使用することによって、より客観的な評価ができる。
または、被験者のある評価対象部位の角層におけるHSP27の発現量を測定し、測定されたHSP27の発現量を、標本集団の同一評価対象部位の角層におけるHSP27の発現量の分布と比較してもよい。標本集団のHSP27の発現量の分布を使用することによって、より客観的な評価ができる。
First, the expression level of HSP27 measured as described above is compared with the expression level of HSP27 in the stratum corneum of healthy skin. In addition, the expression level of HSP27 in the stratum corneum of a subject's evaluation target site is measured, and the measured expression level of HSP27 is compared with the expression level of HSP27 in the stratum corneum of the same evaluation target portion of a person with healthy skin. May be. The expression level of HSP27 in the stratum corneum of people with healthy skin can be evaluated more objectively by using the average value of data from a plurality of people with healthy skin.
Or, measure the expression level of HSP27 in the stratum corneum of a subject site to be evaluated, and compare the measured expression level of HSP27 with the distribution of HSP27 expression level in the stratum corneum of the same target region of the sample population Also good. By using the distribution of the expression level of HSP27 in the sample population, a more objective evaluation can be performed.
このように比較した結果、測定されたHSP27の発現量が、健常肌のHSP27の発現量より有意に大きい場合、または、標本集団のHSP27の発現量分布と比較して、HSP27の発現量が大きいと判断される場合には、ストレスの蓄積が大きいと評価し、さらに敏感肌である可能性が高いと評価することができる。この健常肌の試料は、HSP27を測定する者から採取してもよく、HSP27を測定する者と異なる者から採取してもよい。また、この標本集団は、統計的に有効な規模で、無作為に抽出することが好ましいが、目的に応じて、例えば、性別、年齢別に標本集団を抽出することも好ましい。 As a result of the comparison, when the measured expression level of HSP27 is significantly larger than the expression level of HSP27 in healthy skin, or compared with the expression distribution of HSP27 in the sample population, the expression level of HSP27 is large If it is determined, it can be evaluated that the accumulation of stress is large, and that the possibility of sensitive skin is high. The sample of healthy skin may be collected from a person who measures HSP27, or may be collected from a person different from the person who measures HSP27. In addition, it is preferable to randomly extract the sample group on a statistically effective scale, but it is also preferable to extract the sample group by sex and age according to the purpose.
また、被験者のある評価対象部位の角層におけるHSP27の発現量が、健常肌を有する人の同一評価対象部位の角層におけるHSP27の発現量より有意に大きい場合、または、標本集団の同一評価対象部位の角層におけるHSP27の発現量分布と比較して、HSP27の発現量が大きいと判断される場合にも、ストレスの蓄積が大きいと評価する。またこの数値が顕著に高い場合に敏感肌を有すると評価する。 In addition, when the expression level of HSP27 in the stratum corneum of a subject's evaluation target site is significantly greater than the expression level of HSP27 in the stratum corneum of the same evaluation target region of a person with healthy skin, or the same evaluation target of the sample population Even when it is judged that the expression level of HSP27 is large compared to the distribution of expression level of HSP27 in the stratum corneum at the site, it is evaluated that the accumulation of stress is large. Moreover, when this figure is remarkably high, it evaluates having sensitive skin.
すなわちストレス蓄積が高いと評価する場合、測定されるHSP27の発現量の程度により、ストレスの蓄積度合の程度を評価できる。測定されるHSP27の発現量が健常肌のそれ、または、標本集団の発現量分布と比較して著しく多いと判断される場合は、その肌は、敏感肌の性質を強く示し、健常肌または、標本集団の平均より外界刺激に対する抵抗性が顕著に低く、きわめて容易に皮膚トラブルを生ずる可能性が高い。また、測定されるHSP27の発現量が健常肌のそれと比較して少量だけ増加している場合、または、測定されるHSP27の発現量が標本集団のその発現量分布と比較して、平均値より少し大きいと判断される場合は、その肌は、ストレスの蓄積が標本集団の平均値よりあるが、外界刺激に対する抵抗性が低く皮膚トラブルを生ずるという敏感肌に達していないと評価できる。 That is, when it is evaluated that the stress accumulation is high, the degree of stress accumulation can be evaluated based on the measured expression level of HSP27. If the measured expression level of HSP27 is determined to be significantly higher than that of healthy skin or the expression level distribution of the sample population, the skin strongly exhibits the properties of sensitive skin, Resistance to external stimuli is significantly lower than the average of the sample population, and skin troubles are very likely to occur. In addition, when the measured expression level of HSP27 is increased by a small amount compared to that of healthy skin, or the measured expression level of HSP27 is compared with the expression distribution of the sample population, If it is judged that the skin is a little larger, it can be evaluated that the skin does not reach the sensitive skin where the accumulation of stress is higher than the average value of the sample group but the resistance to external stimuli is low and skin trouble occurs.
以下、具体的な実施例について説明するが、本発明は下記の実施例に限定されるものではない。 Hereinafter, specific examples will be described, but the present invention is not limited to the following examples.
（実験１）紫外線照射による皮膚ストレス蓄積度の試験
（１）紫外線によるストレス負荷
健康な男10名を被験者として試験を実施した。
紫外線照射装置として、Multiple Solar Ultraviolet Simulator Model 601（ソーラーライト社製）を用い、光源に150Wキセノンランプを使用した。被験者の背部皮膚に、色素沈着を誘導するような紅斑が出るように照射強度を調整し、170秒間紫外線（UVB）を照射した。なお、紫外線照射部位と非照射部位とを比較することで、紫外線によるストレス蓄積を確認した。人工紫外線照射前及び照射から5、11、17日目にテープストリッピング法（角層チェッカー：アサヒバイオメッド）により皮膚角層サンプルを採取した。
(Experiment 1) Test of skin stress accumulation degree by ultraviolet irradiation (1) Stress load by ultraviolet ray A test was conducted with 10 healthy men as subjects.
As an ultraviolet irradiation device, Multiple Solar Ultraviolet Simulator Model 601 (manufactured by Solarlight) was used, and a 150 W xenon lamp was used as a light source. The irradiation intensity was adjusted so that erythema that induces pigmentation appeared on the back skin of the subject, and ultraviolet rays (UVB) were irradiated for 170 seconds. In addition, the stress accumulation | storage by ultraviolet rays was confirmed by comparing an ultraviolet irradiation site | part with a non-irradiation site | part. A skin stratum corneum sample was collected by a tape stripping method (horny layer checker: Asahi Biomed) before artificial ultraviolet irradiation and on days 5, 11, and 17 after irradiation.
（２）皮膚角層中タンパク抽出方法
ホモジナイズ用チューブに0.1％SDS/PBS(-)200μlを採取し、これに上記テープストリッピングした角層チェッカーを入れ、ホモジナイゼーション用ペッスルを用いて粘着面を擦ることによりテープ上の角層からタンパクを抽出した。
(2) 0.1% to protein extraction method homogenizing tube in stratum corneum SDS / PBS (-) and 200 mu l was collected, which was charged with the tape stripped stratum corneum checker, the adhesive using a homogenization for pestle Proteins were extracted from the stratum corneum on the tape by rubbing the surface.
（３）皮膚角層タンパクの定量
角層抽出液中に含まれるタンパク量はDC Protein Assay Kit (Bio-Rad製)にて定量した。
(3) Quantification of skin stratum corneum protein The amount of protein contained in the stratum corneum extract was quantified with DC Protein Assay Kit (manufactured by Bio-Rad).
（４）ウエスタンブロット法によるHSP27の定量
1.5μgの角層タンパクをXP PANTERA Gel（5-20％グラジェントゲル：DRC社製）を用いてSDS-PAGEによって分離後、タンパク質をPVDF膜に転写し、StartingBlock Blocking Buffer（Thermo Scientific社製）にてブロッキングを行った。１次抗体（HSP27 Polyclonal Antibody：Stressgen社製）を1000倍希釈、２次抗体（Goat anti-mouse IgG:invitrogen社製）を10000倍希釈で反応させた後、ECLplus（BD Bioscience社製）を用いて、LAS-4000mini（フジフィルム社製）で検出を行った。得られたバンドの発光強度はScience Lab 2004 Multi Gauge（フジフィルム社製）にて数値化を行った。あらかじめ遺伝子組み換えHSP27タンパク質を標準品として作成した検量線により、発光シグナルの強度をHSP27のタンパク質量として読み取って評価を行った。
(4) Quantification of HSP27 by Western blot
After separating 1.5μg of stratum corneum protein by SDS-PAGE using XP PANTERA Gel (5-20% gradient gel: DRC), the protein was transferred to a PVDF membrane, and StartingBlock Blocking Buffer (Thermo Scientific) Blocking was performed. After the primary antibody (HSP27 Polyclonal Antibody: manufactured by Stressgen) was diluted 1000 times and the secondary antibody (Goat anti-mouse IgG: manufactured by invitrogen) was reacted at a dilution of 10,000 times, ECLplus (manufactured by BD Bioscience) was used. Then, detection was performed with LAS-4000mini (Fuji Film). The emission intensity of the obtained band was quantified by Science Lab 2004 Multi Gauge (Fuji Film). Evaluation was performed by reading the intensity of the luminescent signal as the amount of HSP27 protein using a calibration curve prepared in advance using a recombinant HSP27 protein as a standard.
（５）結果
紫外線による刺激（ストレス）を与えた被験者の角層のHSP27量の測定結果を表１、図１にしめす。紫外線が長期間にわたって皮膚にストレスを蓄積させることを確認した。
(5) Results Table 1 and FIG. 1 show the measurement results of the amount of HSP27 in the stratum corneum of the subject given the stimulus (stress) by ultraviolet rays. It was confirmed that ultraviolet rays accumulate stress in the skin over a long period of time.
紫外線照射により皮膚に紅斑が発生し、HSP27は経日的に増加した。これは刺激が持続し、その結果ストレスが蓄積されていったものと評価した。 Erythema developed in the skin by UV irradiation, and HSP27 increased daily. It was evaluated that the stimulation was sustained and stress was accumulated as a result.
（実験２）ソジウムドデシルサルフエート（SDS）による低刺激のストレス蓄積試験
皮膚のHSP27を増加させることが知られている界面活性剤SDSの刺激がストレスとして蓄積されることを確認するため健常肌の男女8名を対象として以下の試験を行った。
(Experiment 2) Low-stimulation stress accumulation test with sodium dodecylsulfate (SDS) Healthy skin to confirm that the stimulation of surfactant SDS, known to increase HSP27 in the skin, accumulates as stress The following tests were conducted on 8 men and women.
（１）ストレスの負荷
1日4回1時間おきに2日間、左前腕内側部を10％SDS水溶液で洗浄して肌荒れ（ストレス状態）を作成した。なお同一人の右前腕を水のみで洗浄し、対照とした。また、左前腕が、水分蒸散量が処理前と比較して上昇していることを確認後に以下の測定を開始した。
(1) Stress load
The inner surface of the left forearm was washed with a 10% SDS aqueous solution four times a day every other hour for 2 days to create rough skin (stress state). The right person's right forearm was washed with water only as a control. Moreover, the following measurement was started after confirming that the amount of moisture transpiration of the left forearm was increased compared with that before the treatment.
（２）試料の採取と水分蒸散量の測定
肌荒れ処理後（0日目）、翌日、3日目、5日目、7日目、14日目に水分蒸散量を測定するとともにテープストリッピング法によって角層サンプルを採取した。なお、14日目においても水分蒸散量の測定値が、肌荒れ処理前の測定値まで戻らなかった場合は21日目、28日目まで測定を延長した。
なお、水分蒸散量の測定は、Courage＆Khazaka社製テバメーターで測定した。角層は角層チェッカー（アサヒバイオメッド）により採取した。
(2) Collection of samples and measurement of moisture transpiration After the rough skin treatment (day 0), the moisture transpiration was measured on the next day, the third day, the fifth day, the seventh day, and the 14th day, and by the tape stripping method. A stratum corneum sample was taken. In addition, when the measured value of the water transpiration amount did not return to the measured value before the rough skin treatment on the 14th day, the measurement was extended to the 21st and 28th days.
The amount of moisture transpiration was measured using a Courage & Khazaka tevameter. The stratum corneum was collected with a stratum corneum checker (Asahi Biomed).
（３）皮膚角層中タンパク抽出方法
実験１と同様に、ホモジナイズ用チューブに0.1％SDS/PBS(-)200μlを採取し、これに上記テープストリッピングした角層チェッカーを入れ、ホモジナイゼーション用ペッスルを用いて粘着面を擦ることによりテープ上の角層からタンパクを抽出した。
(3) Like the stratum corneum in protein extraction methods Experiment 1, the homogenized tube 0.1% SDS / PBS (-) and 200 mu l was collected, which was charged with the tape stripped stratum corneum checker, homogenization Protein was extracted from the stratum corneum on the tape by rubbing the adhesive surface with a pestle.
(４)皮膚角層タンパクの定量
角層抽出液中に含まれるタンパク量はDC Protein Assay Kit（Bio-Rad製）にて定量した。
(4) Quantification of skin stratum corneum protein The amount of protein contained in the stratum corneum extract was quantified with DC Protein Assay Kit (manufactured by Bio-Rad).
（５）ウエスタンブロット法によるHSP27の定量
1.5μgの角層タンパクをXP PANTERA Gel（5-20％グラジェントゲル：DRC社製）を用いてSDS-PAGEによって分離後、タンパク質をPVDF膜に転写し、StartingBlock Blocking Buffer（Thermo Scientific社製）にてブロッキングを行った。1次抗体（Hsp27 PolyclonalAntibody：Stressgen社製）を1000倍希釈、２次抗体（Goat anti-mouse IgG:invitrogen社製）を10000倍希釈で反応させた後、ECLplus（BD Bioscience社製）を用いて、LAS-4000mini（フジフィルム社製）で検出を行った。得られたバンドの発光強度はScience Lab 2004 Multi Gauge（フジフィルム社製）にて数値化を行った。あらかじめ遺伝子組み換えHSP27蛋白質を標準品として作成した検量線により、発光シグナルの強度をHSP27のタンパク質量として読み取って評価を行った。
(5) Quantification of HSP27 by Western blot
After separating 1.5μg of stratum corneum protein by SDS-PAGE using XP PANTERA Gel (5-20% gradient gel: DRC), the protein was transferred to a PVDF membrane, and StartingBlock Blocking Buffer (Thermo Scientific) Blocking was performed. The primary antibody (Hsp27 PolyclonalAntibody: manufactured by Stressgen) was diluted 1000 times and the secondary antibody (Goat anti-mouse IgG: manufactured by invitrogen) was reacted at a 10,000 times dilution, and then ECLplus (manufactured by BD Bioscience) was used. The detection was performed with LAS-4000mini (manufactured by Fujifilm). The emission intensity of the obtained band was quantified by Science Lab 2004 Multi Gauge (Fuji Film). Evaluation was performed by reading the intensity of the luminescence signal as the amount of HSP27 protein using a calibration curve prepared in advance using a recombinant HSP27 protein as a standard.
（６）結果
SDS洗浄による刺激（ストレス）を与えた被験者の角層、並びに対照のHSP27量の測定結果を表２、図２に示す。またあわせて水分蒸散量の変化を示す。
(6) Results
Table 2 and FIG. 2 show the measurement results of the stratum corneum of the subject given the stimulus (stress) by SDS washing and the amount of HSP27 of the control. In addition, the change of moisture transpiration is also shown.
SDS洗浄による皮膚水分の蒸散は、1日目でピークに達し徐々に回復してゆくが、HSP27は14日目まで増加する。これは皮膚のストレスが蓄積されていったものと評価した。 The transpiration of skin moisture by SDS washing reaches its peak on day 1 and gradually recovers, but HSP27 increases until day 14. This was evaluated as the accumulation of skin stress.
（実験３）アトピー性皮膚炎患者のストレス蓄積度の評価
アトピー性皮膚炎患者は、皮膚が常に外界からの刺激を受け続けており、皮膚は常にストレス状態にある。このような状態にある患者の皮膚ストレスの蓄積度を測定した。
（１）皮膚角層採取方法
アトピー性皮膚炎であると診断された女性患者4名の皮膚炎を呈している頚部皮膚より角質チェッカー（2.5cm×2.5cm：アサヒバイオメッド製)を用い、テープストリッピングを行い、角層を採取した。また皮膚炎症状を呈していない上腕部皮膚からも同様にしてサンプリングを行った。
(Experiment 3) Evaluation of Stress Accumulation Level of Atopic Dermatitis Patients In atopic dermatitis patients, the skin is constantly receiving external stimuli, and the skin is always in a stress state. The accumulation degree of the skin stress of the patient in such a state was measured.
(1) Method for collecting skin stratum corneum Using a keratin checker (2.5cm x 2.5cm: manufactured by Asahi Biomed) from cervical skin presenting dermatitis in 4 female patients diagnosed with atopic dermatitis, tapes Tripping was performed and the stratum corneum was collected. Sampling was also carried out in the same manner from the upper arm skin that did not exhibit skin inflammation.
同様にして、健常な女性3名の頚部皮膚、上腕部皮膚からもサンプリングを行い対照とした。 Similarly, sampling was performed from the skin of the neck and upper arm of three healthy women as controls.
（２）皮膚角層中タンパク抽出方法
ホモジナイズ用チューブに1％SDS/PBS(-)200μlを採取し、これに上記テープストリッピングした角層チェッカーを入れ、ホモジナイゼーション用ペッスルで粘着面を擦ることによりテープ上の角層からタンパクを抽出した。
(2) 1% to stratum corneum in protein extraction methods homogenizing tube SDS / PBS (-) and 200 mu l was collected, which was charged with the tape stripped stratum corneum checker, the adhesive surface with homogenization for pestle Proteins were extracted from the stratum corneum on the tape by rubbing.
（３）皮膚角層タンパクの定量
角層抽出液中に含まれるタンパク量はDC Protein Assay Kit（Bio-Rad製）にて定量した。
(3) Quantification of skin stratum corneum protein The amount of protein contained in the stratum corneum extract was quantified with DC Protein Assay Kit (manufactured by Bio-Rad).
（４）ウエスタンブロット法によるHSP27の定量
1.5μgの角層タンパクをXP PANTERA Gel（5-20％グラジェントゲル：DRC社製）を用いてSDS-PAGEによって分離後、タンパク質をPVDF膜に転写し、StartingBlock Blocking Buffer（Thermo Scientific社製）にてブロッキングを行った。１次抗体（Hsp27 Polyclonal Antibody：Stressgen社製）を1000倍希釈、２次抗体（Goat anti-mouse IgG:invitrogen社製）を10000倍希釈で反応させた後、ECLplus（BD Bioscience社製）を用いて、LAS-4000mini（フジフィルム社製）で検出を行った。得られたバンドの発光強度はScience Lab 2004 Multi Gauge（フジフィルム社製）にて数値化を行った。発光シグナルの強度をHSP27の相対量として評価を行った。
(4) Quantification of HSP27 by Western blot
After separating 1.5μg of stratum corneum protein by SDS-PAGE using XP PANTERA Gel (5-20% gradient gel: DRC), the protein was transferred to a PVDF membrane, and StartingBlock Blocking Buffer (Thermo Scientific) Blocking was performed. After the primary antibody (Hsp27 Polyclonal Antibody: manufactured by Stressgen) was diluted 1000 times and the secondary antibody (Goat anti-mouse IgG: manufactured by invitrogen) was reacted at a 10,000 times dilution, ECLplus (manufactured by BD Bioscience) was used. Then, detection was performed with LAS-4000mini (Fuji Film). The emission intensity of the obtained band was quantified by Science Lab 2004 Multi Gauge (Fuji Film). The intensity of the luminescence signal was evaluated as the relative amount of HSP27.
（５）結果
アトピー性皮膚炎患者並びに健常人の皮膚角層のHSP27量の測定結果を表３、図３に示す。
(5) Results Table 3 and FIG. 3 show the measurement results of the amount of HSP27 in the stratum corneum of patients with atopic dermatitis and healthy individuals.
表３、図３から明らかなように、健常人のHSP27は角層中にはほとんど存在しないが、アトピー性皮膚炎患者の患部皮膚からは高濃度にHSP27が検出された。またアトピー性皮膚炎患者の正常皮膚からは、皮膚炎を呈している部位ほどではないが、健常人に比して高濃度にHSP27が検出された。以上の結果から、常時ストレスを受けているアトピー性皮膚炎患者の皮膚はストレスが蓄積されていることがわかる。またアトピー性皮膚炎の患部ではない皮膚も相対的にはHSP27が高いことが確認できた。以上の結果から、敏感肌の指標としてもHSP27を測定することが有用であることが確認できた。 As is apparent from Table 3 and FIG. 3, HSP27 of a healthy person is hardly present in the stratum corneum, but HSP27 was detected at a high concentration from the affected skin of an atopic dermatitis patient. Moreover, HSP27 was detected from normal skin of patients with atopic dermatitis at a higher concentration than that of healthy individuals, although not as much as the site exhibiting dermatitis. From the above results, it can be seen that stress is accumulated in the skin of atopic dermatitis patients who are constantly stressed. It was also confirmed that HSP27 was relatively high in skin that was not affected by atopic dermatitis. From the above results, it was confirmed that it is useful to measure HSP27 as an index of sensitive skin.
（実験３−２）外界のストレスを常に受けている顔面と比較的ストレスを受けていない前腕部や臀部皮膚のHSP27を測定し、皮膚に蓄積されているストレスの程度を評価した。 (Experiment 3-2 ) HSP27 of the face always receiving external stress and the forearm and buttocks skin relatively unstressed was measured to evaluate the degree of stress accumulated in the skin.
試験方法
（１）試料の採取
健常な女性8名より、頬、あご下、前腕、臀部よりテープストリッピング法によって角層サンプルを採取した。1部位より4枚採取した。
Test method (1) Sample collection A stratum corneum sample was collected from 8 healthy women by the tape stripping method from the cheek, under the chin, forearm and buttocks. Four samples were collected from one site.
（２）皮膚角層中タンパク抽出方法
ホモジナイズ用チューブに0.5％chapsバッファー400μlを入れ、これに上記テープストリッピングした角層チェッカーを入れ、ホモジナイゼーションによりテープ上の角層からタンパクを抽出した。1部位から採取した4枚をまとめて1本のチューブで抽出し、1サンプルとした。
(2) Protein extraction method in the skin stratum corneum 400 μl of 0.5% chaps buffer was placed in a homogenization tube, the above-mentioned tape stripped stratum corneum checker was placed, and protein was extracted from the stratum corneum on the tape by homogenization. Four samples collected from one site were extracted together with one tube to make one sample.
(３)皮膚角層タンパクの定量
角層抽出液中に含まれるタンパク量はDC Protein Assay Kit（Bio-Rad製）にて定量した。
(3) Quantification of skin stratum corneum protein The amount of protein contained in the stratum corneum extract was quantified with DC Protein Assay Kit (manufactured by Bio-Rad).
（４）ELISA法によるHSP27の定量
角層抽出液中に含まれるHSP27量はTotal human HSP27 DuoSet ELISA kit（R&D systems製）にて測定した。結果を単位タンパク量当たりのHSP27量として表４、図４に示した。なお表記は平均値±S.E.で示した。
(4) Quantification of HSP27 by ELISA The amount of HSP27 contained in the stratum corneum extract was measured with Total human HSP27 DuoSet ELISA kit (manufactured by R & D systems). The results are shown in Table 4 and FIG. 4 as the amount of HSP27 per unit protein. The notation is shown as mean ± SE.
表４、図４の結果から明らかなように、HSP27の測定結果は日常的なストレスの高い顔面（頬）で特に高く、露出の少ない臀部では顕著に低かった。以上の結果からHSP27は皮膚の日常的なストレスを評価する指標とすることができることが明らかとなった。 As is apparent from the results of Table 4 and FIG. 4, the measurement result of HSP27 was particularly high on the daily stressed face (cheek), and was significantly low on the heel with little exposure. From the above results, it became clear that HSP27 can be used as an index for evaluating daily skin stress.
（実験４）標本集団の角層のHSP27を測定し、ヒストグラムを作成した。 (Experiment 4) HSP27 in the stratum corneum of the sample population was measured and a histogram was created.
試験方法
（１）試験試料の採取
無作為に選抜した女性445名を対象として、頬よりテープストリッピング法によって角層サンプルを採取した。サンプルは1部位より1枚採取した。
Test Method (1) Collection of Test Samples A sample of stratum corneum was collected from the cheeks by tape stripping for 445 randomly selected women. One sample was collected from one site.
（２）皮膚角層中タンパク抽出方法
ホモジナイズ用チューブにRIPAバッファー500μlを入れ、これに上記テープストリッピングした角層チェッカーを入れ、ホモジナイゼーションによりテープ上の角層からタンパクを抽出した（抽出液Ａ）。更に、抽出後の角層チェッカーを、別のホモジナイズ用チューブに移し、1％SDS含有50mMトリス塩酸バッファー500μlを入れ、ホモジナイゼーションによりテープに残った角層からタンパクを抽出した（抽出液Ｂ）。抽出液ＡはHSP27の定量および角層タンパクの定量に用い、抽出液Ｂは角層タンパクの定量に用いた。
(2) Protein extraction method in the skin stratum corneum RIPA buffer 500 μl is put into a homogenization tube, the above-mentioned tape stripping stratum corneum checker is put, and protein is extracted from the stratum corneum on the tape by homogenization (extraction Liquid A). Furthermore, the extracted stratum corneum checker was transferred to another homogenization tube, and 500 μl of 50 mM Tris-HCl buffer containing 1% SDS was added, and proteins were extracted from the stratum corneum remaining on the tape by homogenization (extract B). . Extract A was used for HSP27 and stratum corneum protein, and Extract B was used for stratum corneum protein.
(３)皮膚角層タンパクの定量
角層抽出液中に含まれるタンパク量はBCA protein assay（Pierce製）にて定量した。抽出液Ａと抽出液Ｂのタンパク量の合計をtotal protein量とした。
(3) Quantification of skin stratum corneum protein The amount of protein contained in the stratum corneum extract was quantified by BCA protein assay (Pierce). The total protein amount of Extract A and Extract B was defined as the total protein amount.
（４）ELISA法によるHSP27の定量
角層抽出液中に含まれるHSP27量はTotal human HSP27 DuoSet ELISA kit（R&D systems製）にて測定した。結果は単位タンパク量当たりのHSP27量にて解析した。標本集団のHSP27発現量の度数分布表を表５に、ヒストグラムを図５に示した。
(4) Quantification of HSP27 by ELISA The amount of HSP27 contained in the stratum corneum extract was measured with Total human HSP27 DuoSet ELISA kit (manufactured by R & D systems). The results were analyzed by the amount of HSP27 per unit protein amount. The frequency distribution table of HSP27 expression level of the sample population is shown in Table 5, and the histogram is shown in FIG.
表５、図５に示したとおり、HSP27の発現量は、0.4pg/μg total proteinから50pg/μg total proteinを超える値までの広い範囲で分布している。平均値は15.0pg/μg total proteinであった。この度数分布と比較して、HSP27の発現量が大きいと判断する基準は、自由に設定することが可能であるが、例えば、平均値15.0pg/μg total protein 以上の値をHSP27の発現量が大きいと判断しても良いし、10.0pg/μg total proteinの累積度数が約50％なので、10.0pg/μg total protein を超える値を、HSP27の発現量が大きいと判断しても良いと考える。いずれにしても、HSP27の発現量が大きいほど、ヒト皮膚のストレス蓄積度は大きいと判断できる。
尚、実験４のHSP27の発現量は、実験３の頬部のHSP27の発現量の1/10であるが、これは、実験４のタンパク抽出方法では、HSP27を含むタンパク質を抽出した後に、1％SDS含有50mMトリス塩酸バッファーを用いて角層のタンパク質を抽出しており、実験３と比べて実験４の全タンパク質の検出量が増加したことが原因である。実験３、実験４ともにHSP27を抽出する工程には差がなく、HSP27は溶解性が高いため、当該工程で十分に抽出される。一方、実験４で追加した、SDSによる抽出方法によりタンパク質のほぼ全量を抽出することができるが、当該抽出液中のHSP27を測定することはできない。実験３と実験４の抽出工程が異なるため、実験３と実験４のデータを比較することはできないが、実験３のデータ間の比較、実験４のデータ間の比較は可能である。
As shown in Table 5 and FIG. 5, the expression level of HSP27 is distributed over a wide range from 0.4 pg / μg total protein to a value exceeding 50 pg / μg total protein. The average value was 15.0 pg / μg total protein. The standard for judging that the expression level of HSP27 is large compared to this frequency distribution can be freely set.For example, the expression value of HSP27 is an average value of 15.0 pg / μg total protein or more. Since the cumulative frequency of 10.0 pg / μg total protein may be about 50%, a value exceeding 10.0 pg / μg total protein may be determined to indicate that the expression level of HSP27 is large. In any case, it can be determined that the greater the expression level of HSP27, the greater the degree of stress accumulation in human skin.
The expression level of HSP27 in Experiment 4 is 1/10 of the expression level of HSP27 in the cheek of Experiment 3. This is because the protein extraction method in Experiment 4 extracts 1 protein after HSP27 is extracted. This is because the protein in the stratum corneum was extracted using 50 mM Tris-HCl buffer containing% SDS, and the amount of total protein detected in Experiment 4 increased compared to Experiment 3. There is no difference in the process of extracting HSP27 in both Experiment 3 and Experiment 4, and HSP27 is highly soluble, so it is sufficiently extracted in this process. On the other hand, almost the entire amount of protein can be extracted by the SDS extraction method added in Experiment 4, but HSP27 in the extract cannot be measured. Since the extraction processes of Experiment 3 and Experiment 4 are different, the data of Experiment 3 and Experiment 4 cannot be compared, but the comparison of the data of Experiment 3 and the comparison of the data of Experiment 4 are possible.
（実験５）ヒト皮膚のストレス蓄積度に関するアンケート結果と、HSP27量の対比
実験４の標本集団から、無作為に189名を抽出し、ヒト皮膚のストレス蓄積度に関するアンケートを実施し、その結果とHSP27の発現量を対比した。
(Experiment 5) Comparison of questionnaire results on the degree of stress accumulation in human skin and the amount of HSP27 189 people were randomly selected from the sample population in Experiment 4, and a questionnaire on the degree of stress accumulation in human skin was conducted. The expression level of HSP27 was compared.
（１）肌の敏感性のアンケート
被験者に、被験者の肌が、「非敏感」、「やや敏感」、「敏感」の３種類のうちのいずれであるか選択させた。
下記敏感性の10の要因を被験者に提示し、非敏感（敏感ではない）、やや敏感（やや敏感である）、敏感（敏感である）のいずれに該当するか被験者に自己判断させた。
1 化粧品を使って赤みやかゆみを感じることがある
2 清涼感の高い（スーッした）化粧品が肌にあわないことがある
3 エタノールが入った化粧品が肌にあわないことがある
4 香水や香料が入った化粧品が肌に合わないことがある
5 肌が弱いので化粧品を慎重に選んでいる
6 マッサージなど物理的な刺激によって肌の状態が悪くなることがある
7 普段の生活でも紫外線を浴びたりすると肌が赤くなったり、ヒリヒリしやすい
8 汗をかいた後にかゆみがでることがある
9 化粧品や洗剤があわずに赤みやかゆみがでる
10 化粧品の肌トラブルにより通院した経験がある
「敏感」を選択する者は、ヒト皮膚のストレス蓄積度が高く、「非敏感」を選択する者はヒト皮膚のストレス蓄積度が低いと予想した。
(1) Skin Sensitivity Questionnaire The subject was asked to select whether the subject's skin was one of three types: “non-sensitive”, “somewhat sensitive”, and “sensitive”.
The following 10 factors of sensitivity were presented to the subjects, and the subjects were made to judge whether they were insensitive (not sensitive), slightly sensitive (somewhat sensitive), or sensitive (sensitive).
1 You may feel redness or itching with cosmetics
2 Cosmetic products with a high refreshing sensation may not fit the skin
3 Cosmetics containing ethanol may not match the skin
4 Cosmetics containing perfume or fragrance may not fit the skin
5 Carefully choose cosmetics because of weak skin
6 Physical irritation such as massage may make skin condition worse
7 Even in daily life, exposure to ultraviolet rays makes the skin red and irritated
8 Itching may occur after sweating
9 Redness and itching appear without cosmetics and detergent
10 Those who chose “Sensitive” who had a visit to the hospital due to cosmetic skin problems were expected to have a high degree of stress accumulation in human skin, and those who chose “Insensitive” had a low degree of stress accumulation in human skin.
（２）肌の敏感性のアンケート結果とHSP27量の関係
肌の敏感性のアンケート結果とHSP27量の関係を表６、図６に示す。
(2) Relationship between skin sensitivity questionnaire result and HSP27 amount Table 6 and FIG. 6 show the relationship between the skin sensitivity questionnaire result and HSP27 amount.
（３）屋外活動経験のアンケート
被験者に、屋外活動経験について、「あり」、「なし」のうちでいずれかを選択させた。
屋外活動として、スポーツやガーデニングを例示した。
屋外活動経験「あり」と回答するものはヒト皮膚のストレス蓄積度が高く、「なし」と回答するものはヒト皮膚のストレス蓄積度が低いと予想した。
(3) Outdoor activity experience questionnaire The subjects were asked to select either “Yes” or “No” for outdoor activity experience.
Examples of outdoor activities include sports and gardening.
Those who answered “Yes” in outdoor activities were expected to have high stress accumulation in human skin, and those who answered “None” were expected to have low stress accumulation in human skin.
(４)屋外活動経験のアンケート結果とHSP27量との関係
屋外活動経験のアンケート結果とHSP27量との関係を表７、図７に示す。
( 4 ) Relationship between questionnaire results of outdoor activity experience and HSP27 amount Table 7 and Fig. 7 show the relationship between questionnaire results of outdoor activity experience and HSP27 amount.
Claims (4)
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2012012033A JP5658696B2 (en) | 2011-02-25 | 2012-01-24 | Evaluation method of skin stress accumulation |
| PCT/JP2012/053345 WO2012114932A1 (en) | 2011-02-25 | 2012-02-14 | Method of evaluating degree of skin stress accumulation |
| KR1020137014928A KR20130140069A (en) | 2011-02-25 | 2012-02-14 | Method of evaluating degree of skin stress accumulation |
| CN2012800090971A CN103380375A (en) | 2011-02-25 | 2012-02-14 | Method of evaluating degree of skin stress accumulation |
| TW101105615A TWI580964B (en) | 2011-02-25 | 2012-02-21 | Method for evaluating stress accumulation degree of skin |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2011040355 | 2011-02-25 | ||
| JP2011040355 | 2011-02-25 | ||
| JP2012012033A JP5658696B2 (en) | 2011-02-25 | 2012-01-24 | Evaluation method of skin stress accumulation |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| JP2012189581A JP2012189581A (en) | 2012-10-04 |
| JP2012189581A5 JP2012189581A5 (en) | 2014-09-11 |
| JP5658696B2 true JP5658696B2 (en) | 2015-01-28 |
Family
ID=46720714
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2012012033A Active JP5658696B2 (en) | 2011-02-25 | 2012-01-24 | Evaluation method of skin stress accumulation |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JP5658696B2 (en) |
| KR (1) | KR20130140069A (en) |
| CN (1) | CN103380375A (en) |
| TW (1) | TWI580964B (en) |
| WO (1) | WO2012114932A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5731598B2 (en) * | 2012-11-02 | 2015-06-10 | 株式会社ファンケル | Evaluation method for rough skin |
| CN103808940B (en) * | 2012-11-02 | 2017-06-06 | 株式会社芳珂 | The evaluation method of coarse skin |
| JP5775540B2 (en) * | 2013-02-22 | 2015-09-09 | 株式会社ファンケル | Evaluation method of UV stress applied by UV irradiation of skin |
| JP2015072226A (en) * | 2013-10-03 | 2015-04-16 | 住友ベークライト株式会社 | Inspection method |
| JP5977268B2 (en) * | 2014-01-09 | 2016-08-24 | 株式会社ファンケル | Evaluation method of texture |
| JP6530646B2 (en) * | 2015-06-22 | 2019-06-12 | 株式会社ファンケル | Evaluation method of compressive stress of scalp |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2345330T3 (en) * | 2002-10-02 | 2010-09-21 | The University Of British Columbia | OLIGONUCLEOTIDOS FOR THE TREATMENT OF PROSTATE CANCER AND OTHER CANCERES. |
| JP4469762B2 (en) * | 2005-07-26 | 2010-05-26 | 株式会社資生堂 | Sensitive skin evaluation method and evaluation kit |
| JP5362219B2 (en) * | 2005-08-23 | 2013-12-11 | 株式会社ファンケル | Skin aging marker and its utilization technology |
| JP4954908B2 (en) * | 2008-01-21 | 2012-06-20 | 株式会社 資生堂 | Evaluation method of skin quality using the amount of soluble protein in stratum corneum as an index |
-
2012
- 2012-01-24 JP JP2012012033A patent/JP5658696B2/en active Active
- 2012-02-14 WO PCT/JP2012/053345 patent/WO2012114932A1/en active Application Filing
- 2012-02-14 CN CN2012800090971A patent/CN103380375A/en active Pending
- 2012-02-14 KR KR1020137014928A patent/KR20130140069A/en not_active Application Discontinuation
- 2012-02-21 TW TW101105615A patent/TWI580964B/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| TW201235662A (en) | 2012-09-01 |
| JP2012189581A (en) | 2012-10-04 |
| KR20130140069A (en) | 2013-12-23 |
| WO2012114932A1 (en) | 2012-08-30 |
| TWI580964B (en) | 2017-05-01 |
| CN103380375A (en) | 2013-10-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5658696B2 (en) | Evaluation method of skin stress accumulation | |
| Luebberding et al. | Skin physiology in men and women: in vivo evaluation of 300 people including TEWL, SC hydration, sebum content and skin surface pH | |
| Brach et al. | The reliability and validity of measures of gait variability in community-dwelling older adults | |
| Boireau‐Adamezyk et al. | Age‐dependent changes in stratum corneum barrier function | |
| DeLeo et al. | The effect of race and ethnicity on patch test results | |
| Hadi et al. | The investigation of the skin biophysical measurements focusing on daily activities, skin care habits, and gender differences | |
| Anthonissen et al. | Reliability of Repeated Measurements on Post‐Burn Scars with Corneometer CM 825® | |
| Murdan et al. | An assessment of the human nail plate pH | |
| Lee et al. | Different cosmetic habits can affect the biophysical profile of facial skin: a study of korean and chinese women | |
| JP5065237B2 (en) | Noninvasive evaluation method of local pathology of atopic dermatitis | |
| Zeytun | Demodex (Acari: Demodicidae) infestation in the elderly and its relationship with the skin parameters such as moisture, pH, and temperature: a cross-sectional study. | |
| JP4469762B2 (en) | Sensitive skin evaluation method and evaluation kit | |
| JP5775540B2 (en) | Evaluation method of UV stress applied by UV irradiation of skin | |
| Padmanabhan et al. | Which is more prevalent among the female population-Osteopenia or Osteoporosis? A cross sectional study | |
| Barbosa et al. | Body mass index as discriminator of the lean mass deficit and excess body fat in institutionalized elderly people | |
| Takehara et al. | Association between washing residue on the feet and tinea pedis in diabetic patients | |
| López-Jornet et al. | Study of lip hydration with application of photoprotective lipstick: influence of skin phototype, size of lips, age, sex and smoking habits | |
| JP6017524B2 (en) | Method for testing sensitive skin using stratum corneum | |
| KR101515189B1 (en) | Method for Evaluating Safety of Cosmetics Based on Primary Irritation Index for Human Skin | |
| Huygen et al. | Skin Barrier Function Assessment: Electrical Impedance Spectroscopy Is Less Influenced by Daily Routine Activities Than Transepidermal Water Loss | |
| Farage et al. | Sensitive skin: intrinsic and extrinsic contributors | |
| Dykes | THE MOISTURISING PROPERTIES OF A HEEL BALM IN PATIENTS WITH ROUGH DRY SKIN. | |
| Paul et al. | Detection of skin alterations in mild-to-moderate chronic venous disease using non-invasive techniques | |
| Diehl | The burden of sensitive skin | |
| Hendricks et al. | The Certifed diabetes educator and the Pedorthist working together |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20140728 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20140728 |
|
| A871 | Explanation of circumstances concerning accelerated examination |
Free format text: JAPANESE INTERMEDIATE CODE: A871 Effective date: 20140728 |
|
| A975 | Report on accelerated examination |
Free format text: JAPANESE INTERMEDIATE CODE: A971005 Effective date: 20140814 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20140826 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20141021 |
|
| 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: 20141125 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20141128 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 5658696 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 |