JPH1129442A - Evaluation of hair growing agent - Google Patents
Evaluation of hair growing agentInfo
- Publication number
- JPH1129442A JPH1129442A JP18252897A JP18252897A JPH1129442A JP H1129442 A JPH1129442 A JP H1129442A JP 18252897 A JP18252897 A JP 18252897A JP 18252897 A JP18252897 A JP 18252897A JP H1129442 A JPH1129442 A JP H1129442A
- Authority
- JP
- Japan
- Prior art keywords
- hair
- apoptosis
- epithelial cells
- amount
- evaluation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、育毛剤による、毛
包由来上皮細胞に起こるアポトーシスの抑制作用を評価
することを特徴とする育毛剤の評価方法に関する。TECHNICAL FIELD The present invention relates to a method for evaluating a hair restorer, which comprises evaluating an inhibitory effect of a hair restorer on apoptosis occurring in hair follicle-derived epithelial cells.
【０００２】[0002]
【従来の技術】従来の育毛剤のin vivo 評価系として、
Ｃ３Ｈマウス育毛試験は発毛促進作用のみを評価する方
法として有用であるが、該評価方法を用いて退行期の抑
制又は遅延化を評価することは不可能であった。従っ
て、各種育毛剤が有する退行期の抑制又は遅延化に及ぼ
す作用を詳細に評価するのは困難であり、退行期に関連
する有効な指標を見い出し、測定する必要があった。現
在、ヘアーサイクルの退行期には毛包細胞にアポトーシ
スと呼ばれる細胞死が起こり、このアポトーシスが引金
となって毛成長が止まると考えられている（David Weed
on et al. Acta Dermatovener(Stockholm) 61:335-369,
1981) 。アポトーシスの起こった細胞では、核ＤＮＡが
断片化され、モノヌクレオソーム及びオリゴヌクレオソ
ームが産生される。しかし、ヘアーサイクルにおいて、
モノヌクレオソームやオリゴヌクレオソームの生成量が
測定されたことはなかった。本発明者等は、ヘアーサイ
クルの生化学的指標として、毛包由来細胞中のモノヌク
レオソーム及びオリゴヌクレオソームの総量がＣ３Ｈマ
ウスの退行期間に上昇することを見い出し、モノヌクレ
オソームとオリゴヌクレオソームの総量をＣ３Ｈマウス
のヘアーサイクル退行期において測定し、有用育毛剤の
有する退行期の抑制又は遅延化に及ぼす作用を詳細に評
価することが可能であることを見い出した（特願平８−
６００７５号）。2. Description of the Related Art As a conventional in vivo evaluation system for a hair restorer,
Although the C3H mouse hair growth test is useful as a method for evaluating only the hair growth promoting action, it was impossible to evaluate the suppression or delay of the catagen phase using the evaluation method. Therefore, it is difficult to evaluate in detail the effects of various hair restorers on suppressing or delaying the catagen, and it was necessary to find and measure an effective index relating to the catagen. Currently, it is thought that hair follicle cells undergo cell death called apoptosis during the regression phase of the hair cycle, and this apoptosis triggers hair growth (David Weed
on et al. Acta Dermatovener (Stockholm) 61: 335-369,
1981). In apoptotic cells, nuclear DNA is fragmented, producing mononucleosomes and oligonucleosomes. However, in the hair cycle,
The production of mononucleosomes and oligonucleosomes has never been measured. The present inventors have found that as a biochemical indicator of the hair cycle, the total amount of mononucleosomes and oligonucleosomes in hair follicle-derived cells increases during the regression period of C3H mice. It was measured during the regression phase of the mouse hair cycle, and it was found that the effect of a useful hair restorer on the suppression or delay of the regression phase can be evaluated in detail (Japanese Patent Application No. 8-108).
No. 60075).
【０００３】[0003]
【発明が解決しようとする課題】しかしながら、in viv
o ではアポトーシスの起こる作用メカニズムを詳細に検
討することができないため、in vitroでの評価系が必要
とされる。ところが、invitroで毛包由来上皮細胞をア
ポトーシスに誘導し、そのアポトーシスを抑制する作用
を測定する評価系は未だ無い。本発明の目的は、各種育
毛剤が有する退行期の抑制又は遅延化に及ぼす作用を詳
細に評価するため、退行期に関連する有効な指標を見い
出し測定するin vitroでの評価系を提供することにあ
る。[Problems to be solved by the invention] However, in viv
In the case of o, the mechanism of action of apoptosis cannot be studied in detail, so an in vitro evaluation system is required. However, there is no evaluation system that induces hair follicle-derived epithelial cells to undergo apoptosis in vitro and measures the effect of suppressing the apoptosis. An object of the present invention is to provide an in vitro evaluation system for finding and measuring effective indices related to catagen, in order to evaluate in detail the effects of various hair restorers on suppressing or delaying catagen. It is in.
【０００４】[0004]
【課題を解決するための手段】本発明者等は、上記実情
に鑑み鋭意研究した結果、毛包由来上皮細胞を浮遊培養
することにより、断片化ＤＮＡ量、又はモノヌクレオソ
ームとオリゴヌクレオソームの総量が上昇する、つまり
アポトーシスに誘導できる事を見い出し、浮遊培養した
毛包由来上皮細胞の断片化ＤＮＡ量、又はモノヌクレオ
ソームとオリゴヌクレオソームの総量を測定し、有用育
毛剤の有する退行期の抑制作用を詳細に評価することが
可能である事を見い出した。すなわち、本発明は、育毛
剤のアポトーシス抑制作用を、毛包由来上皮細胞をアポ
トーシスに誘導し、その過程において毛包由来上皮細胞
中の断片化ＤＮＡ量、又はモノヌクレオソームとオリゴ
ヌクレオソームの総量、いずれかを測定することによっ
て評価することを特徴とする育毛剤の評価方法にある。
また、本発明は発毛抑制剤の評価に利用できる評価方法
でもある。Means for Solving the Problems The present inventors have conducted intensive studies in view of the above-mentioned circumstances, and as a result, by floating culture of hair follicle-derived epithelial cells, the amount of fragmented DNA or the total amount of mononucleosomes and oligonucleosomes has been reduced. Increased, that is, induction of apoptosis was found, and the amount of fragmented DNA in hair follicle-derived epithelial cells or the total amount of mononucleosomes and oligonucleosomes in suspension-cultured hair follicles was measured. It was found that it was possible to evaluate. That is, the present invention provides an apoptosis-suppressing effect of a hair-growing agent, inducing hair follicle-derived epithelial cells to undergo apoptosis. The method for evaluating a hair restorer is characterized in that the method is evaluated by measuring the hair growth.
The present invention is also an evaluation method that can be used for evaluating a hair growth inhibitor.
【０００５】[0005]
【発明の実施の形態】本発明者等は、毛包由来上皮細胞
にアポトーシスを誘導し、その抑制作用を毛包由来上皮
細胞中の断片化ＤＮＡ量、又はモノヌクレオソームとオ
リゴヌクレオソームの総量を測定することによって、詳
細な育毛作用を評価することを可能としたが、本発明に
おけるアポトーシスを誘導する方法、断片化ＤＮＡ量、
又はモノヌクレオソームとオリゴヌクレオソームの総量
を測定する方法としては、例えば次の通りである。BEST MODE FOR CARRYING OUT THE INVENTION The present inventors measure the amount of fragmented DNA in hair follicle-derived epithelial cells or the total amount of mononucleosomes and oligonucleosomes in hair follicle-derived epithelial cells by inducing apoptosis in hair follicle-derived epithelial cells. By doing so, it was possible to evaluate the detailed hair growth effect, the method of inducing apoptosis in the present invention, the amount of fragmented DNA,
Alternatively, a method for measuring the total amount of mononucleosomes and oligonucleosomes is as follows, for example.
【０００６】（アポトーシス抑制試験） （１）アポトーシス誘導方法 アガロースをクラボウ社製Ｍ１５４培地に溶解し、オー
トクレーブする。この溶液をプラスチックシャーレに適
量流し込みシャーレをアガロースでコーティングする。
このシャーレにＭ１５４培地に懸濁した毛包由来上皮細
胞を適当量播種し、さらにアポトーシス抑制作用を調べ
たい試料を添加してアガロースコーティングプレート上
で培養（浮遊培養）する。浮遊培養開始２４時間後に細
胞を回収する。(Apoptosis inhibition test) (1) Method for inducing apoptosis Agarose is dissolved in M154 medium manufactured by Kurabo Industries, and autoclaved. An appropriate amount of this solution is poured into a plastic Petri dish, and the Petri dish is coated with agarose.
An appropriate amount of hair follicle-derived epithelial cells suspended in M154 medium is seeded on this dish, and a sample whose apoptosis inhibitory effect is to be examined is further added, followed by culturing (suspension culturing) on an agarose coated plate. The cells are collected 24 hours after the start of the suspension culture.
【０００７】（２）断片化ＤＮＡ量測定方法 回収した細胞に細胞溶解バッファー（１００ｍＭ Ｎａ
Ｃｌ，１０ｍＭ Ｔｒｉｓ−ＨＣｌｐＨ８．０，２５ｍ
Ｍ ＥＤＴＡ，０．５％ＳＤＳ）、さらにその１／１０
量のプロテアーゼＫ水溶液（２ｍｇ／ｍｌ）を加え、細
胞を溶解した。細胞溶解後、タンパクを除く為、等量の
フェノール−クロロホルム−イソアミルアルコール溶液
（２５：２４：１）を加え、攪拌、遠心後、水溶性分画
を取り出す。この操作を２回繰り返して得られた水溶性
分画に２倍量のエタノールを加え、ＤＮＡを析出させ
る。このＤＮＡをＴＥ（１０ｍＭ Ｔｒｉｓ−ＨＣｌｐ
Ｈ７．４，１ｍＭ ＥＤＴＡ）バッファーに溶解し、Ｒ
ＮＡ分解酵素（１０ｍｇ／ｍｌ）を適当量加え、ＲＮＡ
を分解する。その後、１〜数μｇのＤＮＡを１．５％ア
ガロースゲルで電気泳動し、エチジウムブロマイドでＤ
ＮＡを染色する。染色後、ＵＶでＤＮＡを可視化する。
断片化ＤＮＡの量について３段階スコアによって評価を
行う。断片化ＤＮＡ量のスコア評価は、下記表１に示す
評価基準に従って行う。(2) Method for measuring the amount of fragmented DNA [0007] A cell lysis buffer (100 mM Na
Cl, 10 mM Tris-HCl pH 8.0, 25 m
M EDTA, 0.5% SDS), 1/10 of which
An amount of protease K aqueous solution (2 mg / ml) was added to lyse the cells. After cell lysis, an equal amount of a phenol-chloroform-isoamyl alcohol solution (25: 24: 1) is added to remove the protein, and the mixture is stirred and centrifuged, and the water-soluble fraction is taken out. This operation is repeated twice, and twice the amount of ethanol is added to the water-soluble fraction obtained to precipitate DNA. This DNA was converted to TE (10 mM Tris-HClp).
H7.4, 1 mM EDTA) buffer.
Add an appropriate amount of NA-degrading enzyme (10 mg / ml)
Decompose. Thereafter, 1 to several μg of the DNA is electrophoresed on a 1.5% agarose gel, and the DNA is treated with ethidium bromide.
Stain NA. After staining, the DNA is visualized by UV.
The amount of fragmented DNA is evaluated by a three-point score. The score evaluation of the amount of fragmented DNA is performed according to the evaluation criteria shown in Table 1 below.
【０００８】[0008]
【表１】 [Table 1]
【０００９】（３）モノヌクレオソームとオリゴヌクレ
オソームの総量の測定方法 モノヌクレオソームとオリゴヌクレオソームの総量の測
定方法は、細胞死検出キットエライザ（別名：Cell Dea
th Detection ELISA、ベーリンガーマンハイム社製）添
付の使用法に準じて測定する。詳しくは、採取した細胞
にインキュベーションバッファーを添加し、４℃、３０
分間静置する。１５０００ｒｐｍ、１０分間遠心し、上
清の一部を試料とする。予め抗ヒストン抗体をコーティ
ングしたプレートに１０倍希釈した試料を１００μｌ添
加する。室温、９０分静置した後、洗浄溶液で３回プレ
ートを洗う。次に抗ＤＮＡ−パーオキシターゼ抗体溶液
を１００μｌ添加し、室温、９０分静置した後、洗浄溶
液で３回プレートを洗う。最後にパーオキシターゼの基
質（２，２’−アジノ−ジ−［３−エチルベンズチアゾ
リンスルホネート（６）］）溶液を１００μｌ添加し、
４０５ｎｍの波長で吸光度を測定する。尚、モノヌクレ
オソームとオリゴヌクレオソームの総量は一定細胞量の
吸光度で示す。(3) Method for Measuring the Total Amount of Mononucleosomes and Oligonucleosomes The method for measuring the total amount of mononucleosomes and oligonucleosomes is described in Cell Death Detection Kit ELISA (also called Cell Dea
th Detection ELISA, manufactured by Boehringer Mannheim). Specifically, an incubation buffer was added to the collected cells,
Let stand for minutes. Centrifuge at 15000 rpm for 10 minutes, and use a part of the supernatant as a sample. 100 μl of a 10-fold diluted sample is added to a plate previously coated with an anti-histone antibody. After standing at room temperature for 90 minutes, the plate is washed three times with the washing solution. Next, 100 μl of an anti-DNA-peroxidase antibody solution is added, the mixture is left at room temperature for 90 minutes, and the plate is washed three times with a washing solution. Finally, 100 μl of a peroxidase substrate (2,2′-azino-di- [3-ethylbenzthiazoline sulfonate (6)]) solution was added,
The absorbance is measured at a wavelength of 405 nm. The total amount of mononucleosomes and oligonucleosomes is indicated by the absorbance of a fixed amount of cells.
【００１０】本発明で用いる毛包由来上皮細胞は動物の
種類に限定されるものではなく、ヒト、ラット、マウス
などの毛包由来の上皮細胞であれば良い。また本発明に
用いる培地も特に限定されるものではなく毛包由来の上
皮細胞が培養できる培地であればよい。[0010] The hair follicle-derived epithelial cells used in the present invention are not limited to animal types, and may be epithelial cells derived from hair follicles of humans, rats, mice and the like. The medium used in the present invention is not particularly limited as long as the medium can culture epithelial cells derived from hair follicles.
【００１１】[0011]
【実施例】以下に、実施例に基づいて本発明を詳説す
る。尚、これらにより本発明は限定されるものではな
い。DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail based on embodiments. The present invention is not limited by these.
【００１２】実施例１ （１）被験試料 下記表２に示す３種の試料を５０％エタノール中に溶解
し、培地１ｍｌ中に１０μｌ添加し、前記本発明である
毛包由来上皮細胞のアポトーシス抑制試験を実施し、断
片化ＤＮＡ量、及びモノヌクレオソームとオリゴヌクレ
オソームの総量を測定した。測定結果を後記表４に示
す。Example 1 (1) Test Samples Three kinds of samples shown in Table 2 below were dissolved in 50% ethanol, and 10 μl was added to 1 ml of medium to suppress apoptosis of hair follicle-derived epithelial cells of the present invention. Tests were performed to determine the amount of fragmented DNA and the total amount of mononucleosomes and oligonucleosomes. The measurement results are shown in Table 4 below.
【００１３】[0013]
【表２】 [Table 2]
【００１４】（２）評価結果 後記表４に示す如く、本系では、試料３の基剤と比較し
て、試料１、２には、アポトーシスを抑制する効果が認
められた。(2) Evaluation Results As shown in Table 4 below, in the present system, the effects of suppressing apoptosis were observed in Samples 1 and 2 as compared with the base of Sample 3.
【００１５】実施例２ （１）被験試料 下記表３に示す５種の化粧品原料を５０％エタノール中
に溶解し、培地１ｍｌ中に１０μｌ添加し、前記本発明
である毛包由来上皮細胞のアポトーシス抑制試験を実施
し、断片化ＤＮＡ量、及びモノヌクレオソームとオリゴ
ヌクレオソームの総量を測定した。測定結果を後記表４
に示す。Example 2 (1) Test Samples Five kinds of cosmetic raw materials shown in Table 3 below were dissolved in 50% ethanol, and 10 μl was added to 1 ml of a medium. Apoptosis of hair follicle-derived epithelial cells according to the present invention was carried out. An inhibition test was performed to measure the amount of fragmented DNA and the total amount of mononucleosomes and oligonucleosomes. Table 4 below shows the measurement results.
Shown in
【００１６】[0016]
【表３】 [Table 3]
【００１７】（２）評価結果 下記表４に示す如く、試料４〜８にアポトーシスを抑制
する効果が認められた。(2) Evaluation Results As shown in Table 4 below, the effects of suppressing apoptosis were observed in Samples 4 to 8.
【００１８】[0018]
【表４】 [Table 4]
【００１９】[0019]
【発明の効果】以上記載のごとく、本発明は毛包由来上
皮細胞をアポトーシスに誘導し、毛包由来上皮細胞中の
断片化ＤＮＡ量、及び／又はモノヌクレオソームとオリ
ゴヌクレオソームの総量を測定することによって、育毛
剤のアポトーシスの抑制作用をより詳細に評価する方法
を提供し、さらには優れた育毛剤の評価方法を提供する
ことは明らかである。As described above, the present invention induces hair follicle-derived epithelial cells to undergo apoptosis and measures the amount of fragmented DNA in hair follicle-derived epithelial cells and / or the total amount of mononucleosomes and oligonucleosomes. Thus, it is apparent that the present invention provides a method for evaluating the inhibitory action of a hair restorer on apoptosis in more detail, and further provides an excellent method for evaluating a hair restorer.
Claims (1)
し、その過程において毛包由来上皮細胞中の断片化ＤＮ
Ａ量、及び／又はモノヌクレオソームとオリゴヌクレオ
ソームの総量を測定することによって育毛剤のアポトー
シス抑制作用を評価することを特徴とする育毛剤の評価
方法。1. Inducing hair follicle-derived epithelial cells to apoptosis, and in the process, fragmenting DN in hair follicle-derived epithelial cells
A method for evaluating a hair restorer, comprising evaluating the amount of A and / or the total amount of mononucleosomes and oligonucleosomes to evaluate the apoptosis-suppressing action of the hair restorer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18252897A JP3688436B2 (en) | 1997-07-08 | 1997-07-08 | Evaluation method of hair restorer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18252897A JP3688436B2 (en) | 1997-07-08 | 1997-07-08 | Evaluation method of hair restorer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH1129442A true JPH1129442A (en) | 1999-02-02 |
| JP3688436B2 JP3688436B2 (en) | 2005-08-31 |
Family
ID=16119891
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP18252897A Expired - Lifetime JP3688436B2 (en) | 1997-07-08 | 1997-07-08 | Evaluation method of hair restorer |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3688436B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999047924A1 (en) * | 1998-03-18 | 1999-09-23 | Roche Diagnostics Gmbh | Detection of apoptotic products |
| JP2006232828A (en) * | 2005-01-28 | 2006-09-07 | Lion Corp | Hair growing tonic, hair growing tonic composition and method of hair growth and restoration |
-
1997
- 1997-07-08 JP JP18252897A patent/JP3688436B2/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999047924A1 (en) * | 1998-03-18 | 1999-09-23 | Roche Diagnostics Gmbh | Detection of apoptotic products |
| JP2006232828A (en) * | 2005-01-28 | 2006-09-07 | Lion Corp | Hair growing tonic, hair growing tonic composition and method of hair growth and restoration |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3688436B2 (en) | 2005-08-31 |
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