JP2014041043A - Skin condition evaluation method - Google Patents
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本発明は、肌質の評価方法に関する。詳しくは、被験者の肌質を標準的な肌質と比較したり、スキンケアによる肌質の変化を評価できる方法に関する。 The present invention relates to a skin quality evaluation method. Specifically, the present invention relates to a method capable of comparing the skin quality of a subject with standard skin quality or evaluating changes in skin quality due to skin care.
健康的な皮膚を維持するには、皮膚の状態を的確に把握した上で、適切なスキンケアを実践することが何より重要である。そこで、従来、皮膚科医、又は美容専門家による問診等を通じて、皮膚の状態が評価されてきた。また、問診だけでは皮膚状態の客観的な評価が難しい場合もあることから、各種の分析機器を用いた測定により皮膚状態を示すパラメータを算出し、評価する手法も行われている。 In order to maintain healthy skin, it is most important to practice proper skin care after accurately grasping the skin condition. Therefore, conventionally, the state of the skin has been evaluated through an inquiry by a dermatologist or a beauty specialist. In addition, since it is sometimes difficult to objectively evaluate the skin state only by an inquiry, a method for calculating and evaluating a parameter indicating the skin state by measurement using various analytical instruments is also performed.
このような皮膚状態を示すパラメータとしては、例えば、皮膚表面の鋳型（レプリカ）をとり、その表面形態をカメラで画像化して、皮膚表面形状パラメータを算出し、皮紋や皮丘の形態変化を定量的に算出する手法、角層の伝導度測定により角層水分量を測定する方法、経表皮水分蒸散量（TEWL）の測定により角層のバリア機能を評価する方法、不完全な角質細胞の存在比率を測定する方法などが知られている。 As a parameter indicating such a skin state, for example, a skin surface mold (replica) is taken, the surface form is imaged with a camera, a skin surface shape parameter is calculated, and a skin pattern or a skin morphological change is calculated. Quantitative calculation method, method of measuring stratum corneum water content by measuring stratum corneum conductivity, method of evaluating stratum corneum barrier function by measuring transepidermal water transpiration (TEWL), A method for measuring the abundance ratio is known.
さらに、皮膚から採取した角層試料について、炎症性サイトカインとして知られているインターロイキン-1α（以下、「IL-1α」と略称する）の量、及びインターロイキン-1レセプターアンタゴニスト（以下、「IL-1RA」と略称する）の量を測定し、IL-1RA／IL-1αの値を、肌質を評価する指標とする方法も提案されている（特許文献１）。また、このIL-1RA／IL-1αの値を、皮膚疾患の程度を評価する指標とする方法も提案されている（特許文献２）。 Furthermore, for the stratum corneum sample collected from the skin, the amount of interleukin-1α (hereinafter abbreviated as “IL-1α”) known as an inflammatory cytokine, and the interleukin-1 receptor antagonist (hereinafter “IL”) A method of measuring the amount of IL-1RA / IL-1α as an index for evaluating skin quality has also been proposed (Patent Document 1). Further, a method has been proposed in which the IL-1RA / IL-1α value is used as an index for evaluating the degree of skin disease (Patent Document 2).
このように角層試料中に含まれる因子を用いてパラメーターを算出する場合、採取される角層試料の量が評価対象部位や皮膚の状態等により変動することから、採取した角層量あたりの因子量となるように補正する必要がある。そこで、特許文献１及び２では、IL-1RA及びIL-1αを測定する角層抽出試料中の可溶性タンパク質濃度を、ブラッドフォード法で測定し、この可溶性タンパク質の量当たりのIL-1RA／IL-1αの値を、肌質や皮膚疾患の程度を評価する指標としている。 When the parameters are calculated using the factors contained in the stratum corneum sample in this way, since the amount of the stratum corneum sample collected varies depending on the evaluation target site and the skin condition, It is necessary to correct the factor amount. Therefore, in Patent Documents 1 and 2, the soluble protein concentration in the stratum corneum extract sample for measuring IL-1RA and IL-1α is measured by the Bradford method, and IL-1RA / IL− per amount of the soluble protein is measured. The value of 1α is used as an index for evaluating the skin quality and the degree of skin disease.
しかし、特許文献１及び２の方法では、IL-1RA、及びIL-1αの両方の存在量を測定しなければならず、手間がかかる。従って、より簡便で、かつより精度が高い肌質評価方法が求められている。 However, in the methods of Patent Documents 1 and 2, it is necessary to measure the amounts of both IL-1RA and IL-1α, which is troublesome. Accordingly, there is a need for a skin quality evaluation method that is simpler and more accurate.
本発明は、上記従来技術に鑑みてなされたものであり、簡便かつ精度よく肌質を評価できる方法を提供することを課題とする。 This invention is made | formed in view of the said prior art, and makes it a subject to provide the method which can evaluate skin quality simply and accurately.
本発明者らは、前記課題を解決するために鋭意検討した結果、肌質が低下している場合に、角層試料におけるタンパク質の量当たりのインターロイキン-1レセプターアンタゴニスト（IL-1RA）の量が多くなること、及びこのパラメーターを指標にすれば、皮膚の状態を非常に精度良く評価できることを見出した。
本発明は、この知見に基づき完成されたものであり、以下の方法を提供する。
As a result of intensive studies to solve the above problems, the present inventors have found that the amount of interleukin-1 receptor antagonist (IL-1RA) per amount of protein in the stratum corneum sample when skin quality is reduced It was found that the skin condition can be evaluated very accurately by using this parameter as an index.
The present invention has been completed based on this finding, and provides the following method.
項１． 被験者から採取された角層試料におけるタンパク質の量に対するインターロイキン１レセプターアンタゴニストの量の比率を測定する工程を含む、肌質評価方法。
項２． 肌質が炎症の程度である、項１に記載の方法。
項３． 肌質が肌荒れの程度である、項１又は２に記載の方法。
項４． 肌質が、肌老化の程度である、項１〜３のいずれかに記載の方法。
項５． 肌質が、肌の光老化の程度である、項１〜４のいずれかに記載の方法。
項６． タンパク質が分解されたタンパク質である、項１〜５のいずれかに記載の方法。
項７． 分解されたタンパク質がアルカリで分解された分解物である、項６に記載の方法。
項８． 角層試料におけるタンパク質の量を、オルトフタルアルデヒド法により測定する、項１〜７のいずれかに記載の方法。
項９． 角層試料におけるインターロイキン１レセプターアンタゴニストの量を、エンザイムイムノアッセイ法により測定する、項１〜８のいずれかに記載の方法。
項１０． 角層試料におけるタンパク質の量の測定を、インターロイキン１レセプターアンタゴニストの量の測定を終えた角層試料を用いて行う、項１〜９のいずれかに記載の方法。
項１１． 被験者から採取された角層試料におけるタンパク質の量に対するインターロイキン１レセプターアンタゴニストの量の比率を肌質の指標とする、肌質評価方法。
項１２． 被験者から採取された角層試料におけるタンパク質の量に対するインターロイキン１レセプターアンタゴニストの量の比率を測定する工程を含む、肌質試験方法。
Item 1. A skin quality evaluation method comprising a step of measuring a ratio of an amount of an interleukin 1 receptor antagonist to an amount of a protein in a stratum corneum sample collected from a subject.
Item 2. Item 2. The method according to Item 1, wherein the skin quality is the degree of inflammation.
Item 3. Item 3. The method according to Item 1 or 2, wherein the skin quality is a degree of rough skin.
Item 4. Item 4. The method according to any one of Items 1 to 3, wherein the skin quality is the degree of skin aging.
Item 5. Item 5. The method according to any one of Items 1 to 4, wherein the skin quality is the degree of photoaging of the skin.
Item 6. Item 6. The method according to any one of Items 1 to 5, wherein the protein is a degraded protein.
Item 7. Item 7. The method according to Item 6, wherein the degraded protein is a degradation product decomposed with an alkali.
Item 8. Item 8. The method according to any one of Items 1 to 7, wherein the amount of protein in the stratum corneum sample is measured by an orthophthalaldehyde method.
Item 9. Item 9. The method according to any one of Items 1 to 8, wherein the amount of the interleukin 1 receptor antagonist in the stratum corneum sample is measured by an enzyme immunoassay method.
Item 10. Item 10. The method according to any one of Items 1 to 9, wherein the amount of protein in the stratum corneum sample is measured using the stratum corneum sample that has been measured for the amount of interleukin 1 receptor antagonist.
Item 11. A skin quality evaluation method wherein the ratio of the amount of interleukin 1 receptor antagonist to the amount of protein in a stratum corneum sample collected from a subject is used as an index of skin quality.
Item 12. A skin quality test method comprising a step of measuring a ratio of an amount of an interleukin 1 receptor antagonist to an amount of a protein in a stratum corneum sample collected from a subject.
本発明方法は、角層試料のIL-1RAの量、及び角質のタンパク質の量の各測定値を利用するだけの簡便な方法でありながら、高精度で皮膚の状態を評価することができる。従って、本発明により、皮膚の状態を非常に的確に把握することができるようになり、適切なスキンケア方法の選択にも寄与し得る。 The method of the present invention is a simple method that uses only the measured values of IL-1RA and stratum corneum protein in the stratum corneum sample, but can evaluate the skin condition with high accuracy. Therefore, according to the present invention, the skin condition can be grasped very accurately, which can contribute to selection of an appropriate skin care method.
以下、本発明を詳細に説明する。
本発明の方法は、角層試料におけるタンパク質の量当たりのIL-1RA量、即ち、角層試料におけるタンパク質の量に対するIL-1RA量の比率を測定する工程を含む肌質（皮膚状態）評価方法、又は肌質（皮膚状態）試験方法である。
Hereinafter, the present invention will be described in detail.
The method of the present invention is a skin quality (skin condition) evaluation method comprising a step of measuring the amount of IL-1RA per amount of protein in the stratum corneum sample, that is, the ratio of the amount of IL-1RA to the amount of protein in the stratum corneum sample. Or a skin quality (skin condition) test method.
角層試料
角層試料は、表皮角層を含む皮膚組織であればよい。皮膚の部位は特に限定されず、肌質評価が求められる部位（肌質評価対象部位）であればよい。例えば、顔（頬や口唇等）、手、腕、足などの角層試料が好適である。肌の露光部（例えば、顔面の頬部や手の甲）の角層試料は、後述する肌の光老化の程度を評価するのに適している。
角層試料は、例えば、テープストリッピング法、擦過法などの公知の角層採取方法で採取できる。採取した角層試料の水可溶性画分を、IL-1RAの量の測定に供するのが好ましい。
テープストリッピング法は周知の方法であるが、１例を説明すれば、粘着テープの粘着剤層を皮膚の表面に載せ、通常、軽く押さえた後、テープを剥がせばよい。粘着テープの種類は特に限定されず、市販のものを利用すればよい。剥がしたテープには角層組織が付着しているので、テープを細断した後、必要に応じて、水性液に約１〜120分間浸漬して、皮膚組織から水可溶性画分を抽出するのが好ましい。
The stratum corneum sample The stratum corneum sample may be a skin tissue containing the epidermal stratum corneum. The part of the skin is not particularly limited, and may be a part (skin quality evaluation target part) for which skin quality evaluation is required. For example, a stratum corneum sample such as a face (cheek, lips, etc.), hand, arm, or foot is suitable. A stratum corneum sample of an exposed part of the skin (for example, the cheek part of the face or the back of the hand) is suitable for evaluating the degree of photoaging of the skin described later.
The stratum corneum sample can be collected by, for example, a known stratum corneum collection method such as a tape stripping method or a rubbing method. The water-soluble fraction of the collected stratum corneum sample is preferably subjected to measurement of the amount of IL-1RA.
The tape stripping method is a well-known method. To explain one example, the adhesive layer of the adhesive tape is placed on the surface of the skin and usually pressed lightly, and then the tape is peeled off. The kind of adhesive tape is not specifically limited, What is necessary is just to utilize a commercially available thing. Since the stratum corneum tissue is attached to the peeled tape, after chopping the tape, if necessary, it is immersed in an aqueous solution for about 1 to 120 minutes to extract the water-soluble fraction from the skin tissue. Is preferred.
水性液の種類は特に限定されず、水、生理食塩水、リン酸緩衝液、ホウ酸緩衝液、PBS緩衝液、TRIS緩衝液、HEPES緩衝液、CHAPS緩衝液のような緩衝液を使用できる。緩衝液はpH約３〜10のものを使用すればよい。
水性液には、ソルビタン脂肪酸エステル、グリセリン脂肪酸エステル、ポリグリセリン脂肪酸エステル、プロピレングリコール脂肪酸エステル、硬化ヒマシ油誘導体、ポリオキシエチレンソルビタン脂肪酸エステル、ポリオキシエチレンモノヤシ油脂肪酸グリセリル、エトキシ化アルキルフェノール（ポリオキシエチレン（10）オクチルフェニルエーテル、ポリオキシエチレン（10）イソオクチルシクロヘキシルエーテル、ポリオキシエチレン（9）ノニルフェニルエーテル等）のような非イオン性界面活性剤等の界面活性剤を添加してもよく、これにより、一層効率よく水可溶性画分を抽出できる。
The type of the aqueous solution is not particularly limited, and buffers such as water, physiological saline, phosphate buffer, borate buffer, PBS buffer, TRIS buffer, HEPES buffer, and CHAPS buffer can be used. A buffer having a pH of about 3 to 10 may be used.
Aqueous liquids include sorbitan fatty acid esters, glycerin fatty acid esters, polyglycerin fatty acid esters, propylene glycol fatty acid esters, hydrogenated castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene monococonut oil fatty acid glyceryl, ethoxylated alkylphenols (polyoxy Surfactants such as nonionic surfactants such as ethylene (10) octyl phenyl ether, polyoxyethylene (10) isooctyl cyclohexyl ether, polyoxyethylene (9) nonyl phenyl ether) may be added. Thereby, a water-soluble fraction can be extracted more efficiently.
また、浸漬と共に、水性液を撹拌してもよい。また、浸漬、又は撹拌に加えて、約１〜30分間の超音波処理をすることもできる。これらにより、一層効率よく水可溶性画分を抽出できる。
水可溶性画分抽出時の水性液の温度は常温ないしは室温とすることができるが、それより低温（例えば、約0〜5℃）で行えば、水可溶性画分中のタンパク質などの成分の変性を抑えることができる。
Moreover, you may stir an aqueous liquid with immersion. In addition to dipping or stirring, ultrasonic treatment for about 1 to 30 minutes can also be performed. By these, a water-soluble fraction can be extracted more efficiently.
The temperature of the aqueous liquid at the time of water-soluble fraction extraction can be room temperature or room temperature, but if it is performed at a lower temperature (for example, about 0 to 5 ° C.), denaturation of components such as proteins in the water-soluble fraction. Can be suppressed.
また、擦過法などの他の方法により皮膚から採取した角層試料についても、上記と同様にして、水可溶性画分を抽出することができる。 In addition, a water-soluble fraction can be extracted from a stratum corneum sample collected from the skin by other methods such as a scraping method in the same manner as described above.
IL-1RAの量
インターロイキン-1（IL-1）は、主として単球・マクロファージが産生するサイトカインで、等電点の違いによりIL-1αとIL-1βに分類される。生体中のヒトIL-1の約90％はβである。IL-1αとIL-1βの一次構造上の同一性は約26％に過ぎないが、その特異的レセプターは共通であることから、生理機能上の役割は同じと考えられている。なお、生体内では、それぞれ前駆体proIL-1α、proIL-1βとして遺伝子発現し、プロセッシングを受けて活性型IL-1α、IL-1βとなる。これまで、皮膚においても表皮ケラチノサイトでproIL-1α、proIL-1βの発現が確認されているが、α型はproIL-1αからIL-1αに変換されるものの、β型はproIL-1βから活性型IL-1βへと変換する酵素が欠損しているために、プロセッシングを受けずproIL-1βのまま存在するとされてきた。しかし、近年の報告で、角層キモトリプシン様プロテアーゼにより活性型IL-1βへの変換が起こるとも言われているが、テープストリップ法を用いたIL-1関連サイトカインの検出においては、IL-1αが検出可能であったのに対しIL-1βは検出限界以下であったとの報告もなされている。そして、IL-1の最も重要な働きはヘルパーT細胞のIL-2産生を誘導し、IL-2を介してT細胞の分化及び増殖を促進し、生体防御に寄与することである。また、IL-1の発熱作用や滑膜細胞におけるPGE2およびコラゲナーゼの産生誘導、線維芽細胞増殖作用は、生体の炎症過程と密接に関連している。
Amount of IL-1RA Interleukin-1 (IL-1) is a cytokine produced mainly by monocytes and macrophages, and is classified into IL-1α and IL-1β depending on the isoelectric point. About 90% of human IL-1 in the body is β. Although the primary structural identity of IL-1α and IL-1β is only about 26%, their specific receptors are common and thus their physiological roles are considered to be the same. In vivo, genes are expressed as precursors proIL-1α and proIL-1β, respectively, and processed to become active IL-1α and IL-1β. Until now, the expression of proIL-1α and proIL-1β has also been confirmed in the epidermal keratinocytes in the skin, but the α type is converted from proIL-1α to IL-1α, but the β type is activated from proIL-1β Since the enzyme that converts to IL-1β is deficient, it has been assumed that it remains proIL-1β without being processed. However, in recent reports, it is also said that conversion to active IL-1β occurs by stratum corneum chymotrypsin-like protease, but in the detection of IL-1-related cytokines using the tape strip method, IL-1α is It has been reported that IL-1β was below the detection limit while it was detectable. The most important function of IL-1 is to induce IL-2 production of helper T cells, promote differentiation and proliferation of T cells via IL-2, and contribute to biological defense. In addition, IL-1's pyrogenic action, PGE 2 and collagenase production induction in synovial cells, and fibroblast proliferation action are closely related to the inflammatory process in the body.
IL-1レセプターに対するアンタゴニスト（即ち、IL-1RA）の量は、リンパ球増殖反応の阻害等を指標としたバイオアッセイ法、IL-1RAに対する抗体との反応性に基づくエンザイムイムノアッセイ法、ラジオイムノアッセイ法、ウエスタンブロッティング法等の方法を用いて測定できる。操作が簡便で、かつ感度が高い点で、抗体を用いるエンザイムイムノアッセイ法が好ましい。エンザイムイムノアッセイ法は、例えば、市販のキットであるIL-1 Receptor Antagonist, Human, Duoset Kit, DY280（R&D SYSTEMS）などを用いて行うことができる。 The amount of antagonist to IL-1 receptor (ie IL-1RA) is determined by bioassay method using inhibition of lymphocyte proliferation reaction as an index, enzyme immunoassay method based on reactivity with antibody against IL-1RA, radioimmunoassay method It can be measured using a method such as Western blotting. An enzyme immunoassay method using an antibody is preferable in terms of simple operation and high sensitivity. The enzyme immunoassay can be performed using, for example, a commercially available kit such as IL-1 Receptor Antagonist, Human, Duoset Kit, DY280 (R & D SYSTEMS).
IL-1RAの測定値は、通常、測定に供した水可溶性画分に含まれるIL-1RAの存在量に換算したものであるが、濃度であってもよい。即ち、本発明における「IL-1RA量」は、測定に供した水可溶性画分に含まれるIL-1RAの存在量（重量など）、及びIL-1RAの濃度の何れであってもよい。 The measured value of IL-1RA is usually converted to the abundance of IL-1RA contained in the water-soluble fraction subjected to the measurement, but may be a concentration. That is, the “IL-1RA amount” in the present invention may be any of the abundance (weight, etc.) of IL-1RA and the concentration of IL-1RA contained in the water-soluble fraction subjected to the measurement.
タンパク質の量
角層試料に含まれるタンパク質の量は、公知の方法で測定できる。このような公知の方法として、例えば、波長280nmにおけるタンパク質中の芳香族アミノ酸に由来する吸収を測定する紫外線吸収法、色素クーマシーブルーがタンパク質と結合する際の吸光度の変化を測定するブラッドフォード法、フェノール試薬とタンパク質が結合する際の吸光度の変化を測定するローリー法、ビシンコニン酸と銅の錯体がタンパク質と結合する際の吸光度の変化を測定するビシンコニン酸（BCA）法、ペプチド結合と2価銅イオンとの錯体の吸収ピークの吸光度を測定するビューレット法、電気泳動バンドのシグナル強度を測定する電気泳動法、タンパク質と銅イオンとの結合に基づく吸光度を測定する2-D Quant Kitを用いる方法、タンパク質の（又はタンパク質の分解により生成する）第１級アミンとO-フタルアルデヒド（OPA）との結合による蛍光強度を測定するオルトフタルアルデヒド（OPA）法、タンパク質を硫酸で分解することにより窒素をアンモニウムイオンとして定量するケルダール法、タンパク質のジスルフィド結合を還元し、次いで塩酸でアミド結合を加水分解して得られる遊離アミノ酸をニンヒドリンと反応させるニンヒドリン法、タンパク質を燃焼させて得られる窒素ガスをガスクロマトグラフィーにより定量するデュマ法などが挙げられる。
The amount of protein contained in the stratum corneum sample can be measured by a known method. As such known methods, for example, an ultraviolet absorption method for measuring absorption derived from an aromatic amino acid in a protein at a wavelength of 280 nm, a Bradford method for measuring a change in absorbance when the dye Coomassie blue binds to a protein , The Raleigh method for measuring changes in absorbance when a phenol reagent and a protein are bound, the bicinchoninic acid (BCA) method for measuring changes in absorbance when a bicinchoninic acid and copper complex is bound to a protein, peptide binding and divalent Using the Burette method to measure the absorbance of the absorption peak of the complex with copper ion, the electrophoresis method to measure the signal intensity of the electrophoresis band, and the 2-D Quant Kit to measure the absorbance based on the binding between protein and copper ion Method, primary amine and O-phthalaldehyde (OPA) of protein (or produced by protein degradation) Orthophthalaldehyde (OPA) method to measure the fluorescence intensity due to the binding of protein, Kjeldahl method to quantitate nitrogen as ammonium ion by decomposing protein with sulfuric acid, Disulfide bond of protein is reduced, and then amide bond is hydrolyzed with hydrochloric acid Examples include a ninhydrin method in which a free amino acid obtained in this manner is reacted with ninhydrin, a Dumas method in which nitrogen gas obtained by burning protein is quantified by gas chromatography, and the like.
本発明方法では、肌質と、角層試料におけるタンパク質の量に対するIL-1RAの量の比率との相関が良好になる点で、角層試料におけるタンパク質の分解物を定量することが好ましい。例えば、OPA法、ケルダール法、ニンヒドリン法、デュマ法などがタンパク質分解物を定量できる方法であり、OPA法が特に好ましい。タンパク質分解物は、アルカリ又は酸で分解されたものであってもよく、タンパク質分解酵素（例えば、ケラチナーゼ）で分解されたものであってもよく、破砕機等で物理的に分解されたもの等であってもよいが、肌質と、角層試料におけるタンパク質の量に対するIL-1RAの量の比率との相関が一層良好になる点で、アルカリ又は酸で分解されたタンパク質が好ましく、水酸化カリウムなどのアルカリで加水分解した後、塩酸などの酸で中和されたタンパク質分解物がより好ましい。 In the method of the present invention, it is preferable to quantify protein degradation products in the stratum corneum sample in that the correlation between the skin quality and the ratio of the amount of IL-1RA to the amount of protein in the stratum corneum sample becomes good. For example, the OPA method, Kjeldahl method, ninhydrin method, Dumas method and the like are methods capable of quantifying protein degradation products, and the OPA method is particularly preferable. The proteolytic product may be decomposed with an alkali or acid, may be decomposed with a proteolytic enzyme (for example, keratinase), physically decomposed with a crusher, etc. However, a protein decomposed with alkali or acid is preferable in that the correlation between the skin quality and the ratio of the amount of IL-1RA to the amount of protein in the stratum corneum sample is further improved. Proteolytic products that have been hydrolyzed with an alkali such as potassium and then neutralized with an acid such as hydrochloric acid are more preferred.
また、タンパク質、及びタンパク質分解物の何れを定量する場合も、角層試料をそのまま定量に供してもよく、一旦乾燥させた後に定量に供してもよい。好ましくは、角層試料におけるタンパク質又はタンパク質分解物の量の測定は、インターロイキン１レセプターアンタゴニストの量の測定を終えた角層試料（好ましくは、一旦乾燥させた角層試料）を用いて行うのがよい。
本発明における「タンパク質の量」は、測定に供した角層試料に含まれるタンパク質又はタンパク質分解物の存在量（重量など）、及びタンパク質又はタンパク質分解物の濃度の何れであってもよい。測定法によって使用し易い値を用いればよい。
When quantifying either protein or protein degradation product, the stratum corneum sample may be subjected to quantification as it is, or may be subjected to quantification after being once dried. Preferably, the amount of protein or protein degradation product in the stratum corneum sample is measured using a stratum corneum sample (preferably a stratum corneum sample that has been once dried) after measurement of the amount of interleukin 1 receptor antagonist. Is good.
The “amount of protein” in the present invention may be any of the abundance (weight, etc.) of protein or protein degradation product contained in the stratum corneum sample subjected to measurement, and the concentration of protein or protein degradation product. A value that is easy to use depending on the measurement method may be used.
肌質
本発明において「肌質」は、安定肌又は肌荒れの程度を指す。肌荒れは一般に炎症により生じるので、「肌質」は炎症の程度と捉えることもできる。
また、表皮の最外層を構成する角質層は、角質細胞が層をなし、それらの細胞間に細胞間脂質や天然保湿成分が存在している。これにより、外部からの異物の進入を防ぎ、かつ水分の蒸発を防いでいる（バリア機能）。健常な肌（安定肌）では、角質細胞が整然と並び、細胞間に細胞間脂質及び天然保湿物質が存在して、キメが整った状態であるが、肌荒れないしは炎症が起こると、角質細胞間の細胞間脂質の量が少なくなり、肌のキメが乱れて、バリア機能が低下する。後述する実施例に示す通り、角層試料におけるタンパク質の量当たりのIL-1RAの量は、肌のバリア機能の指標となる経表皮水分蒸散量（TEWL）とよく相関する。従って、「肌質」は、皮膚のバリア機能の程度、肌のキメの整い方の程度も包含する。
また、いわゆる敏感肌では、安定肌より、角質間の細胞間脂質の量が少なくなり、肌のキメが乱れて、バリア機能が低下し、刺激の閾値が下がり刺激を感じやすい肌になっている。従って、「肌質」は、敏感肌の程度も包含する。
また、「肌質」は肌の老化の程度も包含し、原因別にみれば、光老化、乾燥老化、酸化老化、皮膚のタンパク質の糖化による老化などが挙げられる。これらは、皮膚内部の微弱炎症の蓄積により進行すると考えられる。
Skin Quality In the present invention, “skin quality” refers to the degree of stable skin or rough skin. Since rough skin is generally caused by inflammation, “skin quality” can also be regarded as the degree of inflammation.
The stratum corneum constituting the outermost layer of the epidermis is composed of stratum corneum, and intercellular lipids and natural moisturizing components are present between these cells. This prevents foreign matter from entering from the outside and prevents water from evaporating (barrier function). In healthy skin (stable skin), keratinocytes are arranged in an orderly manner, intercellular lipids and natural moisturizing substances are present between the cells, and the texture is in order, but when the skin does not get rough or inflammation occurs, The amount of intercellular lipid is reduced, skin texture is disturbed, and the barrier function is lowered. As shown in the Examples described later, the amount of IL-1RA per amount of protein in the stratum corneum sample correlates well with the amount of transepidermal water transpiration (TEWL), which is an index of the skin barrier function. Accordingly, the “skin quality” includes the degree of the barrier function of the skin and the degree of the texture of the skin.
Also, in so-called sensitive skin, the amount of intercellular lipid between the stratum corneum is less than that of stable skin, the skin texture is disturbed, the barrier function is lowered, the stimulation threshold is lowered, and the skin is easy to feel irritation. . Therefore, “skin quality” includes the degree of sensitive skin.
“Skin quality” also includes the degree of skin aging. By cause, photoaging, dry aging, oxidative aging, aging due to glycation of skin proteins, and the like can be mentioned. These are thought to progress due to the accumulation of weak inflammation inside the skin.
本発明における「肌質」は、皮膚医学における「正常」又は「異常」の程度を除く概念である。 “Skin quality” in the present invention is a concept that excludes the degree of “normal” or “abnormal” in dermatology.
工程
本発明の肌質評価方法は、角層試料におけるタンパク質の量に対するIL-1RA量の比率を測定する工程を含む。通常、角層試料におけるタンパク質の量、及びIL-1RAの量をそれぞれ測定すればよいが、タンパク質の量当たりのIL-1RAの量を直接求めてもよい。
本発明方法では、角層試料におけるタンパク質の量に対するIL-1RA量の比率を肌質の指標にする。タンパク質の量当たりのIL-1RAの量の値が高値であることは、肌質が悪いことを意味する。
Process The skin quality evaluation method of the present invention includes a process of measuring the ratio of the amount of IL-1RA to the amount of protein in the stratum corneum sample. Usually, the amount of protein and the amount of IL-1RA in the stratum corneum sample may be measured, respectively, but the amount of IL-1RA per amount of protein may be directly determined.
In the method of the present invention, the ratio of the IL-1RA amount to the protein amount in the stratum corneum sample is used as an index of the skin quality. A high value of the amount of IL-1RA per amount of protein means that the skin quality is poor.
本発明方法は、被験角層試料におけるタンパク質の量に対するIL-1RA量の比率が基準値と比べて高い場合に、基準よりも肌質が悪い（肌荒れの程度が大きい、炎症の程度が大きい、肌のキメが乱れている、肌のバリア機能が低下している、老化の程度が大きい等）と評価、判定、又は判断する工程を含むことができる。
基準値は、被験者の角層試料採取部位と同じ部位から採取した、被験者以外の角層試料の測定値とすることができる。被験者以外の測定値は、被験者について測定するより前に測定した値であってもよく、後に測定した値であってもよい。基準値は、複数人の測定値の平均とするのが好ましく、これにより、被験者の肌質を客観的に評価できる。また、被験者と同年代（好ましくは同性）の複数人の測定値の平均値と、被験者の測定値とを比較することにより、被験者の肌年齢を評価、判定、又は判断することができる。
また、本発明方法では、被験者にスキンケアを施す前の測定値を基準値とし、スキンケアを施した後の測定値をこの基準値と比較することにより、スキンケアが有効であったか否かを評価、判定、又は判断することもできる。具体的には、スキンケアを施す前に角層試料を採取した部位と同一部位又は近傍部位から、スキンケアを施した後に角層試料を採取し、タンパク質量当たりのIL-1RA量の値を測定すればよい。また、皮膚にレーザー治療等を施した被験者において、治療直後の測定値を基準値とし、それから一定期間後の測定値をこの基準値と比較することにより、肌状態の回復度を評価、判定、又は判断することもできる。
When the ratio of the IL-1RA amount to the amount of protein in the test stratum corneum sample is higher than the reference value, the method of the present invention has worse skin quality than the reference (the degree of rough skin is large, the degree of inflammation is large, A step of evaluating, judging or judging that the texture of the skin is disturbed, the barrier function of the skin is lowered, the degree of aging is large, etc.
The reference value can be a measured value of a stratum corneum sample other than the subject collected from the same part as the subject's stratum corneum sample collection part. The measured value other than the subject may be a value measured before the subject is measured, or may be a value measured after the subject. The reference value is preferably an average of the measurement values of a plurality of persons, and thereby the skin quality of the subject can be objectively evaluated. In addition, the skin age of the subject can be evaluated, determined, or determined by comparing the average value of the measured values of a plurality of persons of the same age (preferably the same sex) as the subject and the measured value of the subject.
Further, in the method of the present invention, the measurement value before applying skin care to the subject is used as a reference value, and the measurement value after applying skin care is compared with this reference value to evaluate whether the skin care is effective or not. Or can be determined. Specifically, the stratum corneum sample is collected after applying skin care from the same site as or near the site where the stratum corneum sample was collected before applying skin care, and the IL-1RA amount per protein amount is measured. That's fine. In addition, in subjects subjected to laser treatment or the like on the skin, the measurement value immediately after the treatment is used as a reference value, and then the measurement value after a certain period is compared with this reference value, thereby evaluating and determining the degree of recovery of the skin state. Or it can be judged.
以下に、実施例に基づいて本発明をより詳細に説明するが、本発明はこれらの実施例に限定されない。
（１）角層試料の採取
被験者（20代〜60代の男性43名）の顔面の頬部から、市販のセロハンテープ（登録商標）（ニチバン製）を用いて、角層テープストリッピング法により角層試料を採取した。角層採取面に触れないように細心の注意を払いながら、各テープの大きさが15mm×20mmとなるように余分な幅をカットし、粘着剤層が内側を向くように丸めてマイクロチューブに入れ、角層抽出液（0.05％TritonX−100を含むPBS(-)溶液）400μｌに、テープ全体が浸るようにして浸漬した。
次いで、氷上にて、Twin Mixer TM-282（アズワン製）を用いて２時間アジテーションを行った。その後、各サンプルを遠心分離にかけ（10,000 rpm、５分間）、上清を採取して、角層抽出サンプルとした。この角層抽出サンプルを前述の角層抽出液で３倍に希釈したものを、後述のエンザイムイムノアッセイ法（ELISA法）によるIL-1RAの測定に供した。また、IL-1RA測定後に、角層抽出液で抽出した後のテープに付着した角層サンプルをタンパク質加水分解物の定量に供した。
また、ブランクとして、角層を付着させる前のテープについても同様の操作を行い、ブランクサンプルを得た。
EXAMPLES Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.
(1) Collection of stratum corneum samples Using the commercially available cellophane tape (registered trademark) (manufactured by Nichiban) from the cheeks of the faces of subjects (43 men in their 20s to 60s), the stratum corneum was stripped. A layer sample was taken. While paying close attention not to touch the stratum corneum sampling surface, cut the extra width so that the size of each tape is 15 mm × 20 mm, round it so that the adhesive layer faces inward, and turn it into a microtube It was immersed in the stratum corneum extract (PBS (−) solution containing 0.05% TritonX-100) so that the entire tape was immersed.
Next, agitation was performed on ice using Twin Mixer TM-282 (manufactured by ASONE) for 2 hours. Thereafter, each sample was centrifuged (10,000 rpm, 5 minutes), and the supernatant was collected to obtain a stratum corneum extracted sample. A sample obtained by diluting this stratum corneum extract sample three-fold with the above-mentioned stratum corneum extract was subjected to IL-1RA measurement by an enzyme immunoassay method (ELISA method) described later. In addition, after IL-1RA measurement, the stratum corneum sample attached to the tape after extraction with stratum corneum extract was subjected to protein hydrolyzate quantification.
Moreover, the same operation was performed also about the tape before attaching a stratum corneum as a blank, and the blank sample was obtained.
（２）角層試料におけるIL-1RAの存在量の測定
IL-1RAの測定には、IL-1 Receptor Antagonist, Human, Duoset Kit, DY280（R&D SYSTEMS）を用いた。先ず、コーティング処理として、ELISAプレート（H Type）の各ウェルに10μｇ／mLの捕捉抗体（マウス抗ヒトIL-1RAをPBS（-）を用いて希釈）を100μLずつ加えて、一晩常温にてインキュベートした。翌日、捕捉抗体を捨て、キット付属のWash bufferを１ウェルあたり200μL加え、３回洗浄した。次いで、各ウェルに、キット付属の希釈用試薬（1%BSAを含むPBS）を150μLずつ添加し、室温で１時間インキュベートしてブロッキングを行ない、その後、前記と同様にしてWash bufferで洗浄した。
次いで、ブランクサンプル及び角層抽出サンプルを、別々のウェルに100μLずつ加え、室温で２時間インキュベートして、１次反応を行った後、再度Wash bufferで３回洗浄を行った。続いて、各ウェルに100ng／mLの検出用抗体（ビオチン標識ヤギ抗ヒトIL-1RAを希釈用試薬を用いて希釈）を100μLずつ添加し、室温で２時間インキュベートして２次反応を行い、再度Wash bufferで３回洗浄を行った。続いて、キットに付属のストレプトアビジン-HRP溶液を希釈用溶液を用いて200倍希釈し、各ウェルに100μLずつ添加し、室温にて遮光下で20分間インキュベートを行い、３次反応を行った後に、再度Wash bufferで３回洗浄を行った。
酵素反応として、各ウェルに100μLの基質溶液（Color Reagent AとColor Regent Bを１：１の割合で混合したもの。いずれもR&D SYSTEMS製）を加え、室温にて遮光下で20分間インキュベートを行った。その後、各ウェルに50μLの反応停止液（2N H2SO4）を加えた。最後に、450nmで吸光度の測定を行い、IL-1RAの存在量を算出した。角層抽出サンプルの吸光度からブランクサンプルの吸光度を差し引くことで、補正を行った
(2) Measurement of IL-1RA abundance in stratum corneum samples
IL-1RA was measured using IL-1 Receptor Antagonist, Human, Duoset Kit, DY280 (R & D SYSTEMS). First, as a coating treatment, 100 μL of 10 μg / mL capture antibody (mouse anti-human IL-1RA diluted with PBS (−)) was added to each well of an ELISA plate (H Type) at room temperature overnight. Incubated. On the next day, the capture antibody was discarded, and 200 μL of Wash buffer attached to the kit was added per well and washed three times. Next, 150 μL of the dilution reagent (PBS containing 1% BSA) attached to the kit was added to each well, and the mixture was incubated at room temperature for 1 hour for blocking, and then washed with Wash buffer in the same manner as described above.
Next, 100 μL each of the blank sample and the stratum corneum extraction sample was added to separate wells, incubated at room temperature for 2 hours to perform the primary reaction, and then washed again with Wash buffer three times. Subsequently, 100 μL of 100 ng / mL detection antibody (biotin-labeled goat anti-human IL-1RA diluted with a dilution reagent) was added to each well, and incubated at room temperature for 2 hours to perform a secondary reaction. Washing was performed again with Wash buffer three times. Subsequently, the streptavidin-HRP solution supplied with the kit was diluted 200-fold with a diluting solution, 100 μL was added to each well, and incubated at room temperature for 20 minutes under light shielding to carry out a tertiary reaction. Thereafter, washing was performed again with Wash buffer three times.
As an enzymatic reaction, add 100 μL of the substrate solution (Color Reagent A and Color Regent B mixed at a ratio of 1: 1 in each well. Both are manufactured by R & D SYSTEMS) and incubate for 20 minutes at room temperature in the dark. It was. Thereafter, 50 μL of a reaction stop solution (2N H 2 SO 4 ) was added to each well. Finally, the absorbance was measured at 450 nm, and the abundance of IL-1RA was calculated. Correction was performed by subtracting the absorbance of the blank sample from the absorbance of the stratum corneum extraction sample.
（３）角層試料におけるタンパク質分解物の存在量の測定
前述のIL-1RA量の測定を終えた角層サンプルを、乾燥させ、タンパク質分解物の定量に供した。
先ず、検量線作成用にタンパク質分解物の存在量を測定したいテープと同じ大きさのテープを1.5mLエッペンチューブに入れた。10mg／mLのBSA水溶液を、8M KOHを用いて0、100、200μｇ／mLに希釈調製し、このBSA溶液を各々400μLずつエッペンチューブに添加した。
続いて、IL-1RA量の測定を終えた角層サンプルの乾燥物にも、各々400μLの8M KOHを添加した。
検量線作成用の各濃度のBSA溶液及び角層サンプル溶液を、100℃で8時間にわたり加水分解した。この際、エッペンチューブの蓋が飛ぶ恐れがあったため、エッペンチューブの蓋と加熱機の蓋をしっかりと閉じるように注意を払った。加熱後、各エッペンチューブを氷上に移し、サンプルが十分に冷えたことを確認してから、遠心分離（10,000rpm、5min）にかけた。続いて5M HClを600μLずつ加え、Voltexにより十分に攪拌を行って中和させた。その後、遠心分離（10,000rpm、5min）を行った。
角層サンプル溶液、及び検量線作成用BSA溶液サンプルより採取した上清を96ウェルプレートの各ウェルに100μLずつ入れ、それにオルトフタルアルデヒド（OPA）試薬を50μLずつ加えて、各タンパク質分解物のアミノ基を検出した。その後、励起波長：365nm、蛍光波長：450nmで蛍光の測定を行い、角層試料におけるタンパク質分解物の存在量を算出した。
(3) Measurement of abundance of protein degradation product in stratum corneum sample The stratum corneum sample after the measurement of the amount of IL-1RA described above was dried and subjected to quantification of the protein degradation product.
First, a tape having the same size as the tape whose protein degradation product is to be measured for preparing a calibration curve was placed in a 1.5 mL Eppendorf tube. A 10 mg / mL BSA aqueous solution was diluted to 0, 100, and 200 μg / mL using 8M KOH, and 400 μL each of this BSA solution was added to an Eppendorf tube.
Subsequently, 400 μL of 8M KOH was added to each dried stratum corneum sample after measurement of the amount of IL-1RA.
Each concentration of BSA solution and stratum corneum sample solution for preparing a calibration curve were hydrolyzed at 100 ° C. for 8 hours. At this time, since there was a possibility that the lid of the Eppendorf tube would fly, care was taken to close the lid of the Eppendorf tube and the lid of the heater firmly. After heating, each Eppendorf tube was transferred to ice, and after confirming that the sample was sufficiently cooled, it was centrifuged (10,000 rpm, 5 min). Subsequently, 5 μM HCl was added in an amount of 600 μL, and the mixture was neutralized by thoroughly stirring with Voltex. Thereafter, centrifugation (10,000 rpm, 5 min) was performed.
Add 100 μL of the supernatant collected from the stratum corneum sample solution and the BSA solution sample for preparing the calibration curve to each well of a 96-well plate, add 50 μL of orthophthalaldehyde (OPA) reagent to each well, and add amino acid of each protein degradation product. The group was detected. Thereafter, fluorescence was measured at an excitation wavelength of 365 nm and a fluorescence wavelength of 450 nm, and the abundance of protein degradation products in the stratum corneum sample was calculated.
（４）経表皮水分蒸散量（TEWL）の測定
上記のようにして角層試料を採取した各被験者の角層採取部位の付近について、経表皮水分蒸散量の測定を行った。具体的には、被験者の顔面（頬部）のTEWLを測定した。TEWLは、Vapo Scan AS-VT100RS（株式会社アサヒテクノラボ製）を、添付の取扱説明書に順じて使用することで測定を行った。
(4) Measurement of transepidermal water transpiration (TEWL) The transepidermal water transpiration was measured in the vicinity of the stratum corneum sampling site of each subject who collected the stratum corneum sample as described above. Specifically, the TEWL of the subject's face (cheek) was measured. TEWL measured by using Vapo Scan AS-VT100RS (manufactured by Asahi Technolab Co., Ltd.) according to the attached instruction manual.
（５）角層試料におけるＩＬ−１ＲＡ／角層タンパク質分解物の値と、TEWL値との相関評価
被験者（43名）について、角層試料中タンパク質の量当りのIL-1RAの量を横軸に、そしてTEWLの値を縦軸にとり、プロットしたグラフを図１に示す。このグラフから、相関係数のｔ検定で有意性の検定を行ったところ、p<0.01で有意であることが判明した（y=0.2153x-10.441、r=0.405）。
(5) Correlation between the value of IL-1RA / keratin layer protein degradation product in the stratum corneum and the TEWL value For 43 subjects, the amount of IL-1RA per amount of protein in the stratum corneum is plotted on the horizontal axis. FIG. 1 shows a graph plotted with the value of TEWL on the vertical axis. From this graph, when the significance test was performed by the t-test of the correlation coefficient, it was found that p <0.01 was significant (y = 0.2153x-10.441, r = 0.405).
TEWLは、肌荒れ時のバリア機能を表す皮膚生理パラメーターであり、TEWL値が高いことは表皮からの水分蒸散量が多く、角層のバリア機能が低下していることを示す。即ち、老化や紫外線等の原因により皮膚が炎症を起こし、肌が荒れてバリア機能が低下していることを、このTEWL値から読み取ることができる。本試験例では露光部である顔面の頬部からの角層試料を採取して試験を行っており、本実施例で測定されたTEWL値は光老化を一因とした皮膚の炎症症状を示していると考えられる。
図１から明らかなように、角層試料におけるタンパク質の量に対するIL-1RAの量の比率は、TEWL値と高い正の相関関係を示した。このことは、角層試料におけるタンパク質の量に対するIL-1RAの量の比率を指標とする本発明の肌質評価方法が、皮膚の状態（肌質）を非常に高い精度で評価できる方法であることを示している。
TEWL is a skin physiological parameter that represents a barrier function during rough skin, and a high TEWL value indicates that the amount of water transpiration from the epidermis is large and the barrier function of the stratum corneum is reduced. That is, it can be read from this TEWL value that the skin is inflamed due to aging, ultraviolet rays, etc., and the skin is rough and the barrier function is lowered. In this test example, the stratum corneum sample from the facial cheek part which is the exposed part was collected and tested, and the TEWL value measured in this example shows the inflammatory symptoms of the skin due to photoaging. It is thought that.
As is clear from FIG. 1, the ratio of the amount of IL-1RA to the amount of protein in the stratum corneum sample showed a high positive correlation with the TEWL value. This is a method by which the skin quality evaluation method of the present invention using the ratio of the amount of IL-1RA to the amount of protein in the stratum corneum as an index can evaluate the skin state (skin quality) with very high accuracy. It is shown that.
本発明方法は、簡便な方法でありながら、高精度で皮膚の状態を評価して適切なスキンケア方法の選択にも寄与し得るため、実用性の高い方法である。 Although the method of the present invention is a simple method, it can contribute to the selection of an appropriate skin care method by evaluating the skin condition with high accuracy, and is therefore a highly practical method.
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