JP6530646B2 - Evaluation method of compressive stress of scalp - Google Patents
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Description
本発明は、頭皮の圧縮応力の評価方法に関する。 The present invention relates to a method for evaluating scalp compressive stress.
女性の毛髪は４０代を過ぎると、頭頂部の薄毛化が進行することが確認されている（非特許文献１）。また、頭皮に弾力性があるほど、発毛状態が良くなると言われており、頭皮をつまんだときの圧縮応力が大きいほど、発毛状態が良いと考えられる。
ところで、頭皮の圧縮応力を評価するためには、専用機器を用いた手間のかかる測定が必要であるが、店舗や通信販売において、多数の被験者に対して頭皮ケアのアドバイスを行うために、前記の測定を行うことは困難である。そこで、被験者を拘束せず、簡便に頭皮の圧縮応力を評価できる方法が求められている。
It has been confirmed that thinning of hair on the top of the head progresses when the hair of women passes their 40s (Non-patent Document 1). Also, it is said that the more elastic the scalp, the better the hair growth state, and the greater the compressive stress when pinching the scalp, the better the hair growth state.
By the way, in order to evaluate the compressive stress of the scalp, it is necessary to take time-consuming measurement using a dedicated device, but in order to give advice on scalp care to a large number of subjects in a store or mail order, It is difficult to measure the Therefore, there is a need for a method that can easily evaluate the compressive stress of the scalp without constraining the subject.
本出願人は、ＨＳＰ−２７タンパク質がアトピー性皮膚炎マーカーとして有用であること（特許文献１）、皮膚の紫外線ストレス蓄積の指標となること（特許文献２）を見出した。 The applicant has found that the HSP-27 protein is useful as an atopic dermatitis marker (Patent Document 1) and that it is an indicator of UV stress accumulation in the skin (Patent Document 2).
本発明は、被験者に負担を与えることなく、頭皮をつまんだときの圧縮応力を評価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。 An object of the present invention is to provide a novel evaluation method using a biochemical index which can evaluate compressive stress when pinching the scalp without giving a burden to a subject.
本発明は以下の構成である。
（１）皮膚角層のＨＳＰ−２７の発現量を測定し、その多寡を指標とする、頭皮をつまんだときの圧縮応力の評価方法。
（２）被験者の評価対象部位の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ＨＳＰ−２７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ＨＳＰ−２７の発現量をあらかじめ測定した無作為に選択された標本集団の前記評価対象部位の角層のＨＳＰ−２７の発現量分布と比較する比較工程と、を備えた（１）に記載の評価方法。
（３）被験者の評価対象部位が頭皮または顔面である（２）に記載の評価方法。
The present invention has the following configuration.
(1) A method of evaluating compressive stress when pinching the scalp by measuring the expression level of HSP-27 in the horny layer of skin and using the amount as an index.
(2) a collecting step of collecting the stratum corneum of the evaluation target site of the subject, a measuring step of measuring the expression level of HSP-27 in the horny layer collected in the collecting step, and the HSP- measured in the measuring step A comparison step of comparing the expression level of 27 with the expression level distribution of HSP-27 in the stratum corneum of the evaluation target site of the randomly selected sample group in which the expression level of 27 was measured in advance (1) .
(3) The evaluation method as described in (2) whose evaluation object site | part of a test subject is a scalp or a face.
本発明の評価方法によれば、頭皮の圧縮応力を、角層におけるＨＳＰ−２７の発現量を測定することで評価できる。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、被験者が自宅で行うことが可能であるため、通信販売の手法によって頭皮の圧縮応力の評価結果を提供することが可能となる。 According to the evaluation method of the present invention, the compressive stress of the scalp can be evaluated by measuring the expression level of HSP-27 in the stratum corneum. Furthermore, since the evaluation method of the present invention can be performed at home by the subject because the evaluation sample is collected by a simple operation method such as a tape stripping method, evaluation results of compression stress of the scalp by a method of mail order It becomes possible to offer.
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ＨＳＰ−２７の存在量を指標とすることを特徴としている。
Hereinafter, the present invention will be described in detail.
The evaluation method of the present invention is characterized in that the amount of HSP-27 in the stratum corneum is used as an index.
ＨＳＰは細胞の加熱に反応して発現することが知られており、この発現は細菌感染や炎症、エタノール、活性酸素、重金属、紫外線、飢餓、低酸素状態などの細胞に対する様々なストレスにより誘導されることが知られている。核内タンパク質である熱ショック転写因子（ＨＳＦ）はＤＮＡ上の熱ショックエレメント（ＨＳＥ）に結合することによりＨＳＰの発現を制御する転写因子として働くが、熱ストレスによりＨＳＦが誘導される詳細な機構については十分に明らかにされてはいない。 HSP is known to be expressed in response to heating of cells, and this expression is induced by bacterial infection and inflammation, and various stresses on cells such as ethanol, reactive oxygen species, heavy metals, ultraviolet light, starvation and hypoxia. It is known that The heat shock transcription factor (HSF), a nuclear protein, acts as a transcription factor to control the expression of HSP by binding to heat shock element (HSE) on DNA, but the detailed mechanism by which HSF is induced by heat stress Is not well understood.
本発明では、角層という用語を使用する。角層は、皮膚の一番上にある組織をいう。角層は、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。 In the present invention, the term stratum corneum is used. The stratum corneum refers to the tissue at the top of the skin. The stratum corneum has the function of protecting the skin from foreign bodies and stimuli 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、主な部位としては頭皮、顔面が挙げられ、従来の方法に従い、これらの部位から皮膚由来の角層を得ることができる。しかし、外科的に皮膚を摘出する等の方法は、被験者に負担を与えるため、テープストリッピング、擦過等の非侵襲的に角層を得る方法を用いる。 The site to be evaluated in the present invention may include any site from which the stratum corneum can be obtained, but the main sites include the scalp and the face, and according to the conventional method, the skin origin is derived from these sites The stratum corneum can be obtained. However, methods such as surgical removal of the skin use a method of non-invasively obtaining a stratum corneum such as tape stripping and abrasion in order to put a burden on the subject.
こうして用意した各試料におけるＨＳＰ−２７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、ＨＳＰ−２７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 HSP-27 in each sample thus prepared can be measured by a conventionally known method. For example, methods such as enzyme immunoassay based on reaction with an antibody against HSP-27, radioimmunoassay, western blotting and the like can be used.
各試料からＨＳＰ−２７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し、抽出後、速やかに測定する。 The soluble fraction of HSP-27 is prepared from each sample via a biochemical method known per se, such as freezing and thawing method, sonication method, homogenate method and the like, and it is measured immediately after extraction.
頭皮をつまんだときの圧縮応力が大きい被験者の角層中のＨＳＰ−２７の発現量は高い傾向にあるため、角層におけるＨＳＰ−２７の発現量の多寡を指標として、簡便に頭皮をつまんだときの圧縮応力を評価できる。
本発明に用いる角層は、テープストリッピング等により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。このため、被験者に負担を与えることなく、粘弾性計等の特別な機器を使用することなく、被験者の頭皮の圧縮応力を評価することができる。特に、被験者の頭皮に、より適した化粧品や洗浄剤を簡単に選択することができ、それによって、脱毛の改善が期待できる。
Because the expression level of HSP-27 in the stratum corneum of subjects with large compressive stress when pinching the scalp tends to be high, the scalp is easily pinched using the expression level of HSP-27 in the stratum corneum as an index It is possible to evaluate the compressive stress of time.
The stratum corneum used in the present invention can be collected by a simple method in which only the surface layer portion of the stratum corneum is collected with horny tape by tape stripping or the like. Therefore, the compressive stress of the subject's scalp can be evaluated without giving a burden to the subject and without using a special instrument such as a viscometer. In particular, cosmetic and cleaning agents more suitable for the subject's scalp can be easily selected, whereby improvement in hair loss can be expected.
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ＨＳＰ−２７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ＨＳＰ−２７の発現量を標本集団の角層におけるＨＳＰ−２７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 comprises a collecting step of collecting the stratum corneum, a measuring step of measuring the expression level of HSP-27 in the stratum corneum collected in the collecting step, and the HSP-27 measured in the measuring step. It comprises a comparison step of comparing the expression level with the distribution of HSP-27 expression levels in the stratum corneum of the sample population.
The evaluation method will be described below.
まず、被験者のある評価対象部位の角層におけるＨＳＰ−２７の発現量を測定し、測定されたＨＳＰ−２７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ＨＳＰ−２７の発現量の分布と比較し、ＨＳＰ−２７の発現量の多寡を判定する。標本集団のＨＳＰ−２７の発現量の分布を使用することによって、客観的にＨＳＰ−２７の発現量の多寡を評価できる。 First, the expression level of HSP-27 in the stratum corneum of a certain evaluation target site of the subject is measured, and the measured expression level of HSP-27 is the expression level of HSP-27 in the stratum corneum of the same evaluation target site of the sample population The amount of HSP-27 expression is determined by comparison with the distribution of By using the distribution of the expression level of HSP-27 in the sample population, it is possible to objectively evaluate the level of expression level of HSP-27.
このように比較した結果、測定されたＨＳＰ−２７の発現量が、標本集団のＨＳＰ−２７の発現量分布と比較して、ＨＳＰ−２７の発現量が大きいと評価される場合には、発毛状態が良く、頭皮の圧縮応力が大きいと評価できる。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 this comparison, when the measured expression level of HSP-27 is evaluated to be larger than the expression level distribution of HSP-27 in the sample population, the expression level of HSP-27 is higher. It can be evaluated that the hair condition is good and the compressive stress of the scalp is large. The sample group is preferably extracted at random on a statistically effective scale, but it is also preferable to extract the sample group according to the purpose, for example, gender and age.
角層の採取部位は身体のどこの部位でも構わないが、顔面又は頭皮が好ましく、頭皮が特に好ましい。 Although the collection site of the stratum corneum may be anywhere on the body, the face or scalp is preferable, and the scalp is particularly preferable.
本発明を試験例、実施例によって説明する。なお本発明は下記の実施例に限定されるものではない。 The invention is illustrated by test examples and examples. The present invention is not limited to the following examples.
試験例１
角層中のＨＳＰ−２７の発現量と頭皮をつまんだときの圧縮応力との関係を調べた。
（１）試験方法
＜角層採取＞
被験者：髪の悩み（薄毛、軟毛化）が顕在化するとされる40歳以上の女性 74名（40代（56名）、50代（15名）、60代（3名））
角層採取部位：頭皮
角層採取方法：頭皮を70%アルコールで拭いた。毛を分けて地肌を露出させ、角層採取用テープ（アサヒバイオメッド社製 角層チェッカー ２．２ｃｍ×２．２ｃｍ）を縦に３分割し、３分割した部分それぞれに、３カ所の地肌の角層を付着させた。さらに、その角層採取用テープを横に３分割し、３分割した部分それぞれに、新たな３カ所の地肌の角層を付着させた。すなわち、被験部位は０．７ｃｍ×２．２ｃｍの範囲を６カ所であり、３カ所は角層採取用テープに直接付着させ、残りの３カ所は先の３カ所の角層が付着したテープに方向を変えて重ねて付着させた。
Test Example 1
The relationship between the expression level of HSP-27 in the stratum corneum and the compressive stress when the scalp was pinched was investigated.
(1) Test method <corneal layer collection>
Subject: 74 women (40s (56), 50s (15), 60s (3)) Women over the age of 40 who appear to have hair problems (thin hair, softened hair)
Corneal layer collection site: scalp Radius layer collecting method: The scalp was wiped with 70% alcohol. The hair is divided to expose the ground surface, and the stratum corneum collection tape (Asahi Bio Med Co., Ltd. stratum stratum checker 2.2 cm × 2.2 cm) is vertically divided into three, and in each of the three divided parts, three ground surface The stratum corneum was allowed to adhere. Further, the stratum corneum collecting tape was horizontally divided into three, and three new ground stratum corneum were attached to each of the three divided portions. In other words, the test site is 0.7 cm × 2.2 cm in six locations, three locations are directly attached to the stratum corneum collection tape, and the remaining three locations are on the tape with the previous three stratum corneum attached. The direction was changed, and it was made to attach again.
＜角層中ＨＳＰ−２７測定＞
ガラスビーズとT-PERバッファー（Thermo scientific社）500μlの入ったチューブに角層を採取した角層採取用テープを入れ、25分ボルテックスミキサーにて振とうし、角層タンパクを抽出した。各サンプルのタンパク濃度はBCA protein Assay Kit (Thermo Scientific社)で測定した。測定には角層サンプルを10μlに reagentA: reagentB=50:1で混和した液200μlを加え、60℃30分でインキュベーションしたのち、562nmの吸光度で測定した。同時にウシ血清アルブミン（BSA）で検量線を作成した。この検量線と吸光度の値からタンパク濃度を算出した。
角層抽出液中に含まれるHSP27濃度はHuman HSP27 DuoSet (R&D systems社)を用いて定量した。
角層抽出液中に含まれるHSP27濃度を角層抽出液に含まれるタンパク濃度で割って、角層中のHSP27発現量とした。
<Measurement of HSP-27 in stratum corneum>
The stratum corneum-collecting tape from which the stratum corneum was collected was placed in a tube containing glass beads and 500 μl of T-PER buffer (Thermo scientific) and shaken with a vortex mixer for 25 minutes to extract stratum corneum proteins. The protein concentration of each sample was measured by BCA protein Assay Kit (Thermo Scientific). For measurement, 200 μl of a mixture of the stratum corneum sample in 10 μl of reagent A: reagent B = 50: 1 was added, incubated at 60 ° C. for 30 minutes, and then measured by absorbance at 562 nm. At the same time, a standard curve was prepared with bovine serum albumin (BSA). The protein concentration was calculated from the calibration curve and the absorbance value.
The concentration of HSP27 contained in the stratum corneum extract was quantified using Human HSP27 DuoSet (R & D systems).
The HSP27 concentration contained in the stratum corneum extract was divided by the protein concentration contained in the stratum corneum extract to obtain the amount of HSP27 expression in the stratum corneum.
＜頭皮をつまんだときの圧縮応力の測定＞
頭皮粘弾性計（スキングリップメータ アサヒバイオメッド社製 AS-GP1）を用いて、頭皮をつまんで幅７ｍｍから６ｍｍに圧縮し、その際の圧縮応力を測定した。
<Measurement of compressive stress when pinching the scalp>
The scalp was pinched and compressed to a width of 7 mm to 6 mm using a scalp viscometer (skin grip meter Asahi BioMed AS-GP1), and the compressive stress at that time was measured.
（２）結果
角層中のＨＳＰ−２７の発現量と頭皮をつまんだときの圧縮応力の関係を図１に示す。角層中のＨＳＰ−２７の発現量と頭皮をつまんだときの圧縮応力は正の相関関係を示した（Ｒ２＝０．０５２８）。今回の被験者を標本集団とすれば、頭皮をつまんだときの圧縮応力は、[頭皮をつまんだときの圧縮応力]＝０．５７９１×[角層中のＨＳＰ−２７の発現量]+ １２５．３の計算式で評価できる。
(2) Results The relationship between the expression level of HSP-27 in the stratum corneum and the compressive stress when the scalp is pinched is shown in FIG. The expression level of HSP-27 in the stratum corneum and the compressive stress when pinching the scalp showed a positive correlation (R 2 = 0.0528). Assuming that the subject is a sample group, the compressive stress when pinching the scalp is [Compressive stress when pinching the scalp] = 0.5791 × [the amount of HSP-27 expressed in stratum corneum] + 125. It can be evaluated by the formula of 3.
Claims (2)
A collecting step of collecting the stratum corneum of the subject's scalp , a measuring step of measuring the expression level of HSP-27 in the horny layer collected in the collecting step, and an expression amount of HSP-27 measured in the measuring step The evaluation method according to claim 1, further comprising: a comparison step of comparing the expression level distribution of HSP-27 in the stratum corneum of the scalp of the randomly selected sample group measured in advance.
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