JP2017009426A - Method of evaluating hair growth condition and compressive stress on scalp - Google Patents
Method of evaluating hair growth condition and compressive stress on scalp Download PDFInfo
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本発明は、発毛の状態及び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 the state of hair growth and the compressive stress of the scalp.
女性の毛髪は４０代を過ぎると、１つの毛群から生えている平均毛髪数の減少が認められ、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、発毛の状態及び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った。１つの毛群から生えている平均毛髪数を測定するためには、ビデオマイクロスコープにて頭皮画像を取り込み、毛髪の数、毛群の数を数え上げて記録することが必要である。また、頭皮の圧縮応力を測定するためには、専用の粘弾性計を用いる必要があった。
しかしながら、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、簡便に発毛の状態及び頭皮の圧縮応力を評価できる方法が求められている。
When female hair is over 40 years of age, a decrease in the average number of hairs growing from one hair group is observed, and it has been confirmed that thinning of the head of the head proceeds (Non-patent Document 1). Moreover, it is said that the more elastic the scalp, the better the hair growth state. The greater the compressive stress when pinching the scalp, the better the hair growth state. By the way, in order to evaluate the state of hair growth and the compressive stress of the scalp, time-consuming measurement using a dedicated device is necessary. In order to measure the average number of hairs growing from one hair group, it is necessary to capture a scalp image with a video microscope and count and record the number of hairs and the number of hair groups. Moreover, in order to measure the compressive stress of the scalp, it was necessary to use a special viscoelasticity meter.
However, it is difficult to perform the above measurement in order to give scalp care advice to a large number of subjects in stores and mail-order sales. Therefore, there is a need for a method that can easily evaluate the hair growth state and the compressive stress of the scalp without restraining the subject.
本出願人は、抗酸化作用を有するＤＪ−１タンパク質（別名ＰＡＲＫ-７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）、皮膚のストレス蓄積の指標となること（特許文献２）、紫外線障害の抵抗性の指標となること（特許文献３）を見出した。 The applicant of the present invention is that an anti-oxidative DJ-1 protein (also known as PARK-7 protein) is useful as an atopic dermatitis marker (Patent Document 1), and serves as an index of skin stress accumulation (Patent Document) 2) It has been found that it becomes an index of resistance to UV damage (Patent Document 3).
本発明は、被験者に負担を与えることなく、発毛の状態及び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。 This invention makes it a subject to provide the novel evaluation method using the biochemical parameter | index which can evaluate the state of hair growth and the compressive stress when pinching the scalp, without giving a test subject a burden.
本発明は以下の構成である。
（１）皮膚角層のＤＪ−１の発現量を測定し、その多寡を指標とする、発毛の状態の評価方法。
（２）発毛の状態が頭髪の毛群あたりの平均毛髪数である（１）に記載の方法。
（３）皮膚角層のＤＪ−１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。
（４）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ＤＪ−１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ＤＪ−１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ＤＪ−１の発現量分布と比較する比較工程と、を備えた（１）〜（３）のいづれかに記載の評価方法。
（５）被験者の評価対象部位が頭皮または顔面である（４）に記載の評価方法。
The present invention has the following configuration.
(1) A method for evaluating the state of hair growth, wherein the expression level of DJ-1 in the skin stratum corneum is measured, and the amount of the expression is used as an index.
(2) The method according to (1), wherein the state of hair growth is the average number of hairs per hair group.
(3) A method for evaluating compressive stress when the scalp is pinched, by measuring the expression level of DJ-1 in the stratum corneum and using the amount of the expression as an index.
(4) A sampling step of sampling the stratum corneum of the subject's evaluation target, a measuring step of measuring the expression level of DJ-1 in the stratum corneum sampled in the sampling step, and DJ- measured in the measuring step A comparison step of comparing the expression level distribution of DJ-1 in the stratum corneum of the evaluation target site of a randomly selected sample population in which the expression level of 1 is measured in advance. The evaluation method described in any one.
(5) The evaluation method according to (4), wherein the subject to be evaluated is a scalp or a face.
本発明の評価方法によれば、発毛の状態、具体的には、頭髪の毛群あたりの平均毛髪数を、角層におけるＤＪ−１の発現量を測定することで評価できる。また、頭皮の圧縮応力を、角層におけるＤＪ−１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による画像解析を必要としない発毛状態の評価が可能になる。テープストリッピング法による角層採取は、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発毛の状態、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 state of hair growth, specifically, the average number of hairs per head hair group can be evaluated by measuring the expression level of DJ-1 in the stratum corneum. Moreover, the compressive stress of the scalp can be evaluated by measuring the expression level of DJ-1 in the stratum corneum. 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. Moreover, since it is a biochemical test method and the same result is obtained regardless of who measures it, it is possible to evaluate the hair growth state without the need for image analysis by a conventional expert. Since stratum corneum sampling by the tape stripping method can be performed at home by a subject, it is possible to provide evaluation results of hair growth state and scalp compressive stress by a mail order 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ＤＪ−１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 abundance of DJ-1 in the stratum corneum is used as an index.
ＤＪ−１は、分子質量２１ｋＤａの細胞内タンパク質である。パーキンソン病に関係したタンパク質として同定されたが、その後皮膚の創傷治癒過程での再上皮化（ｒｅ−ｅｐｉｔｈｅｌｉａｌｉｚａｔｉｏｎ）に関与することが明らかになった。また、その立体構造からプロテアーゼとしても働いている可能性が示唆されている。 DJ-1 is an intracellular protein having a molecular mass of 21 kDa. Although identified as a protein associated with Parkinson's disease, it was subsequently shown to be involved in re-epithelialization during skin wound healing. In addition, the three-dimensional structure suggests the possibility of working as a protease.
本発明では、毛群、毛群あたりの平均毛髪数という用語を使用する。 In the present invention, the terms hair group, average number of hairs per hair group are used.
毛群とは、いくつかの毛嚢が集まって形成している１つの毛髪グループのことをいう。 A hair group refers to one hair group formed by collecting several hair follicles.
毛群あたりの平均毛髪数は、頭皮の観察画像中の毛髪の数／毛群の数で表される。 The average number of hairs per hair group is represented by the number of hairs in the observation image of the scalp / number of hair groups.
また、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。 The stratum corneum refers to the tissue on top of the skin. The stratum corneum 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 can include any site as long as the stratum corneum can be obtained, but examples of the main site include the scalp and the face. The stratum corneum can be obtained. However, methods such as surgically removing the skin use a method of obtaining the stratum corneum non-invasively, such as tape stripping or rubbing, in order to put a burden on the subject.
こうして用意した各試料におけるＤＪ−１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、ＤＪ−１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 DJ-1 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 reaction with an antibody against DJ-1 can be used.
各試料からＤＪ−１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。 A soluble fraction is prepared from each sample by biochemical methods known per se, such as freeze-thaw method, ultrasonic crushing method, homogenate method, etc., and measured immediately after extraction.
毛群あたりの平均毛髪数が大きい、発毛状態の良い被験者の角層中のＤＪ−１の発現量は高い傾向にあるため、角層におけるＤＪ−１の発現量の多寡を指標として、簡便に発毛状態（毛群あたりの平均毛髪数）を評価できる。また、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ＤＪ−１の発現量は高い傾向にあるため、角層におけるＤＪ−１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、被験者の発毛状態や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。
Since the expression level of DJ-1 in the stratum corneum of a subject having a large average hair number per hair group and a good hair growth state tends to be high, the expression level of DJ-1 in the stratum corneum is easily used as an index. The hair growth state (average number of hairs per hair group) can be evaluated. In addition, since the expression level of DJ-1 in the stratum corneum of a subject having a large compressive stress when pinching the scalp tends to be high, the scalp can be easily removed using the expression level of DJ-1 in the stratum corneum as an index. The compressive stress when pinched can be evaluated.
The stratum corneum used in the present invention can be collected by a simple method of collecting only the surface layer portion of the stratum corneum with a keratin tape by tape stripping or the like. For this reason, the subject's hair growth state and the compressive stress of the subject's scalp can be evaluated without imposing a burden on the subject. In particular, it is possible to easily select cosmetics and cleaning agents that are more suitable for the scalp of the subject, thereby improving hair loss.
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ＤＪ−１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ＤＪ−１の発現量を標本集団の角層におけるＤＪ−１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 measuring step of measuring the expression level of DJ-1 in the stratum corneum sampled in the sampling step, and the DJ-1 measured in the measuring step It comprises a comparison step of comparing the expression level with the distribution of the expression level of DJ-1 in the stratum corneum of the sample population.
This 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ＤＪ−１の発現量を測定し、測定されたＤＪ−１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ＤＪ−１の発現量の分布と比較し、ＤＪ−１の発現量の多寡を判定する。標本集団のＤＪ−１の発現量の分布を使用することによって、客観的にＤＪ−１の発現量の多寡を評価できる。 First, the expression level of DJ-1 in the stratum corneum of a part to be evaluated of a subject is measured, and the expression level of DJ-1 thus measured is expressed as the expression level of DJ-1 in the stratum corneum of the same evaluation target part of the sample population. And the amount of expression of DJ-1 is determined. By using the distribution of the expression level of DJ-1 in the sample population, the amount of expression level of DJ-1 can be objectively evaluated.
このように比較した結果、測定されたＤＪ−１の発現量が、標本集団のＤＪ−１の発現量分布と比較して、ＤＪ−１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 the comparison, if the measured expression level of DJ-1 is evaluated to be larger than the expression level distribution of DJ-1 in the sample population, It can be evaluated that the hair state is good and that the scalp has a large compressive stress. The sample group is preferably extracted at random on a statistically effective scale, but it is also preferable to extract the sample group by sex and age according to the purpose.
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。 The stratum corneum may be collected anywhere on the body, but 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 present invention will be described with reference to test examples and examples. The present invention is not limited to the following examples.
試験例１
角層中のＤＪ−１の発現量と毛群あたりの平均毛髪数との関係を調べた。
（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35歳以上の女性 103名（30代（29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 DJ-1 in the stratum corneum and the average number of hairs per hair group was examined.
(1) Test method <Extracting stratum corneum>
Subject: Women over 35 years old who are considered to have hair problems (thinning, softening) 103 (30 (29), 40 (56), 50 (15), 60 (3) Name))
Corneal layer collection site: scalp Corneal layer collection method: The scalp was wiped with 70% alcohol. Divide the hair to expose the background, and divide the stratum corneum sampling tape (Asahi BioMed's stratum corneum checker 2.2 cm x 2.2 cm) vertically into three parts, and then add three parts of the scalp to each of the three parts. A stratum corneum was deposited. Further, the stratum corneum sampling tape was divided into three lateral parts, and three new stratum corneums were applied to each of the three parts. That is, the test site is a 0.7 cm × 2.2 cm range of 6 locations, 3 locations are directly attached to the stratum corneum sampling tape, and the remaining 3 locations are tapes to which the previous 3 stratum corneums are attached. The direction was changed and it piled up and adhered.
＜角層中ＤＪ−１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ＤＪ−１濃度はHuman Park7/ＤＪ−１ DuoSet (R&D systems社)を用いて定量した。
角層抽出液中に含まれるＤＪ−１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ＤＪ−１発現量とした。
<DJ-1 measurement in stratum corneum>
The stratum corneum collection tape which collected the stratum corneum was put into a tube containing glass beads and 500 μl of T-PER buffer (Thermo scientific), and the stratum corneum protein was extracted by shaking with a vortex mixer for 25 minutes. The protein concentration of each sample was measured with BCA protein Assay Kit (Thermo Scientific). For the measurement, 200 μl of a stratum corneum sample mixed with 10 μl of reagentA: reagentB = 50: 1 was added, incubated at 60 ° C. for 30 minutes, and then measured by absorbance at 562 nm. At the same time, a calibration curve was prepared with bovine serum albumin (BSA). The protein concentration was calculated from the calibration curve and absorbance values.
The concentration of DJ-1 contained in the stratum corneum extract was quantified using Human Park7 / DJ-1 DuoSet (R & D systems).
The DJ-1 concentration contained in the stratum corneum extract was divided by the protein concentration contained in the stratum corneum extract to obtain the expression level of DJ-1 in the stratum corneum.
＜毛群あたりの平均毛髪数の測定＞
マイクロスコープ（スカラ社 ビデオルーペVL-7EX）を用いて、観察視野 縦７ｍｍ×横９．６ｍｍ ６７．２ｍｍ２の頭皮画像を記録した。記録した頭皮画像を、縦２×横２の４分野に分割し、毛髪数を数え易い２分野を選択し、その２分野（３３．６ｍｍ２）について毛髪の数と毛群の数を目視で計測した。
毛群あたりの平均毛髪数は[毛髪の数]／[毛群の数]で求めた。
<Measurement of the average number of hairs per hair group>
Using a microscope (SCARA Video Loupe VL-7EX), a scalp image having an observation visual field length of 7 mm × width of 9.6 mm, 67.2 mm 2 was recorded. The recorded scalp image is divided into 4 fields of 2x2 in length, and 2 fields where the number of hairs is easy to count are selected, and the number of hairs and the number of hair groups are visually checked for the 2 fields (33.6mm 2 ). Measured.
The average number of hairs per hair group was determined by [number of hairs] / [number of hair groups].
（２）結果
角層中のＤＪ−１の発現量と毛群あたりの平均毛髪数の関係を図１に示す。角層中のＤＪ−１の発現量と毛群あたりの平均毛髪数は正の相関関係を示した（Ｒ２＝０．０５７６）。今回の被験者を標本集団とすれば、毛群あたりの平均毛髪数は、[毛群あたりの平均毛髪数]＝０．０６０９×[角層中のＤＪ−１の発現量]+ １．６３１８の計算式で評価できる。
(2) Results FIG. 1 shows the relationship between the expression level of DJ-1 in the stratum corneum and the average number of hairs per hair group. The expression level of DJ-1 in the stratum corneum and the average number of hairs per hair group showed a positive correlation (R 2 = 0.0576). If the subject is the sample population, the average number of hairs per hair group is [average number of hairs per hair group] = 0.0609 × [expression amount of DJ-1 in the stratum corneum] +1.6318. Can be evaluated by calculation formula.
試験例２
角層中のＤＪ−１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：頬
角層採取方法：洗顔し、頬の角層を角層採取用テープ（アサヒバイオメッド社製 角層チェッカー ２．２ｃｍ×２．２ｃｍ）に付着させ、採取した。
Test example 2
The relationship between the expression level of DJ-1 in the stratum corneum and the compressive stress when pinching the scalp was examined.
(1) Test method <Extracting stratum corneum>
Subjects: Women over 40 years old who are considered to have hair problems (thinning, softening) 74 (40 (56), 50 (15), 60 (3))
Corneal layer collection site: cheek Corneal layer collection method: Face wash, the stratum corneum of the cheek was attached to a tape for stratum corneum collection (corneal layer checker 2.2 cm × 2.2 cm, manufactured by Asahi Biomed) and collected.
＜角層中ＤＪ−１測定＞
試験例１と同様に、角層中のＤＪ−１の発現量を測定した。
<DJ-1 measurement in stratum corneum>
In the same manner as in Test Example 1, the expression level of DJ-1 in the stratum corneum was measured.
＜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>
Using a scalp viscoelasticity meter (skin grip meter AS-GP1 manufactured by Asahi Biomed), the scalp was pinched and compressed from 7 mm to 6 mm in width, and the compression stress at that time was measured.
（２）結果
角層中のＤＪ−１の発現量と頭皮をつまんだときの圧縮応力の関係を図２に示す。角層中のＤＪ−１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＝０．０６５２）。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]＝１７．７４４×[角層中のＤＪ−１の発現量]+ ９４．３５３の計算式で評価できる。
(2) Results FIG. 2 shows the relationship between the expression level of DJ-1 in the stratum corneum and the compressive stress when pinching the scalp. The expression level of DJ-1 in the stratum corneum and the compressive stress when pinching the scalp showed a positive correlation (R 2 = 0.0652). If the subject is the sample population, the compressive stress when pinching the scalp is [compressive stress when pinching the scalp] = 17.744 × [expression amount of DJ-1 in the stratum corneum] +94. It can be evaluated by the calculation formula 353.
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