JP3688436B2 - Evaluation method of hair restorer - Google Patents
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- JP3688436B2 JP3688436B2 JP18252897A JP18252897A JP3688436B2 JP 3688436 B2 JP3688436 B2 JP 3688436B2 JP 18252897 A JP18252897 A JP 18252897A JP 18252897 A JP18252897 A JP 18252897A JP 3688436 B2 JP3688436 B2 JP 3688436B2
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- hair
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Description
【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。
【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 Weedon 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）。
【０００３】
【発明が解決しようとする課題】
しかしながら、in vivo 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、in vitro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。
【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。
【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。
【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。
【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 ＮａＣｌ，１０ｍＭ Ｔｒｉｓ−ＨＣｌｐＨ８．０，２５ｍＭ 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−ＨＣｌｐＨ７．４，１ｍＭ ＥＤＴＡ）バッファーに溶解し、ＲＮＡ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ＤＮＡ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。
【０００８】
【表１】
（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 Death 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。
【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。
【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。
【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。
【００１３】
【表２】
（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。
【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。
【００１６】
【表３】
（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。
【００１８】
【表４】
【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。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for evaluating a hair-growth agent characterized by evaluating the inhibitory action of apoptosis occurring in hair follicle-derived epithelial cells by the hair-growth agent.
[0002]
[Prior art]
As an in vivo evaluation system for conventional hair restorers, the C3H mouse hair growth test is useful as a method for evaluating only the hair growth-promoting action, but it is not possible to evaluate inhibition or delay of the regression phase using this evaluation method. It was possible. Therefore, it is difficult to evaluate in detail the effect of various hair restorers on the suppression or delay of the regression phase, and it is necessary to find and measure an effective index related to the regression phase. Currently, it is thought that hair follicle cells die during the regression phase of the hair cycle, and this apoptosis is triggered to stop hair growth (David Weedon et al. Acta Dermatovener (Stockholm) 61 : 335-369,1981). In apoptotic cells, nuclear DNA is fragmented and mononucleosomes and oligonucleosomes are produced. However, the amount of mononucleosomes and oligonucleosomes produced in the hair cycle has never been measured.
The present inventors have found that the total amount of mononucleosomes and oligonucleosomes in hair follicle-derived cells increases during the regression period of C3H mice as a biochemical indicator of the hair cycle, and the total amount of mononucleosomes and oligonucleosomes is determined as C3H. It has been found that it is possible to evaluate in detail the effect of the useful hair restorer on the suppression or delay of the regression phase, as measured in the hair cycle regression phase of mice (Japanese Patent Application No. 8-60075).
[0003]
[Problems to be solved by the invention]
However, since in vivo the mechanism of apoptosis cannot be examined in detail, an in vitro evaluation system is required. However, there is still no evaluation system for inducing apoptosis of hair follicle-derived epithelial cells in vitro and measuring the effect of suppressing the apoptosis in vitro.
An object of the present invention is to provide an in vitro evaluation system for finding and measuring an effective index related to the regression phase in order to evaluate in detail the effect of various hair restorers on the suppression or delay of the regression phase. It is in.
[0004]
[Means for Solving the Problems]
As a result of intensive studies in view of the above circumstances, the present inventors have found that the amount of fragmented DNA or the total amount of mononucleosomes and oligonucleosomes can be increased, that is, induced by apoptosis, by suspension culture of hair follicle-derived epithelial cells. The amount of fragmented DNA in hair follicle-derived epithelial cells cultured in suspension or the total amount of mononucleosomes and oligonucleosomes can be measured, and the inhibitory effect on the regression phase of useful hair restorers can be evaluated in detail. I found out. That is, the present invention induces the apoptosis-suppressing action of the hair growth agent to induce apoptosis in hair follicle-derived epithelial cells, and in the process, either the amount of fragmented DNA in the hair follicle-derived epithelial cells or the total amount of mononucleosomes and oligonucleosomes, It is in the evaluation method of the hair restorer characterized by evaluating by measuring. Moreover, this invention is also an evaluation method which can be utilized for evaluation of a hair growth inhibitor.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
The present inventors induced apoptosis in hair follicle-derived epithelial cells, and measured the amount of fragmented DNA in the hair follicle-derived epithelial cells or the total amount of mononucleosomes and oligonucleosomes for their inhibitory action. Although it was possible to evaluate the action, examples of the method for inducing apoptosis, the amount of fragmented DNA, or the total amount of mononucleosomes and oligonucleosomes in the present invention are as follows.
[0006]
(Apoptosis suppression test)
(1) Apoptosis induction method 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 in this petri dish, and a sample to be further examined for apoptosis-suppressing action is added and cultured on an agarose-coated plate (floating culture). Cells are collected 24 hours after the start of suspension culture.
[0007]
(2) Method for measuring the amount of fragmented DNA A cell lysis buffer (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 25 mM EDTA, 0.5% SDS) and a 1/10 amount of protease K aqueous solution (2 mg / ml) were added to the collected cells. ml) was added to lyse the cells. After cell lysis, in order to remove protein, an equal amount of a phenol-chloroform-isoamyl alcohol solution (25: 24: 1) is added, stirred and centrifuged, and then the water-soluble fraction is taken out. Two times the amount of ethanol is added to the water-soluble fraction obtained by repeating this operation twice to precipitate the DNA. This DNA is dissolved in TE (10 mM Tris-HCl pH 7.4, 1 mM EDTA) buffer, and an appropriate amount of RNase (10 mg / ml) is added to degrade RNA. Thereafter, 1 to several μg of DNA is electrophoresed on a 1.5% agarose gel, and the DNA is stained with ethidium bromide. After staining, the DNA is visualized with UV. The amount of fragmented DNA is evaluated by a three-level score. The score evaluation of the amount of fragmented DNA is performed according to the evaluation criteria shown in Table 1 below.
[0008]
[Table 1]
(3) Method for measuring the total amount of mononucleosomes and oligonucleosomes The method for measuring the total amount of mononucleosomes and oligonucleosomes is based on the method attached to the Cell Death Detection Kit Eliser (also known as Cell Death Detection ELISA, manufactured by Boehringer Mannheim). taking measurement. Specifically, an incubation buffer is added to the collected cells and left at 4 ° C. for 30 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 anti-histone antibody. After standing at room temperature for 90 minutes, the plate is washed 3 times with a washing solution. Next, 100 μl of an anti-DNA-peroxidase antibody solution is added and the mixture is allowed to stand at room temperature for 90 minutes, and then 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 is added, and the absorbance is measured at a wavelength of 405 nm. The total amount of mononucleosomes and oligonucleosomes is indicated by the absorbance of a certain amount of cells.
[0010]
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 it can culture epithelial cells derived from hair follicles.
[0011]
【Example】
Hereinafter, the present invention will be described in detail based on examples. The present invention is not limited by these.
[0012]
Example 1
(1) Test sample Three types of samples shown in Table 2 below were dissolved in 50% ethanol, 10 μl was added to 1 ml of medium, and the apoptosis inhibition test of hair follicle-derived epithelial cells according to the present invention was carried out. The amount of conjugated DNA and the total amount of mononucleosomes and oligonucleosomes were measured. The measurement results are shown in Table 4 below.
[0013]
[Table 2]
(2) Evaluation Results As shown in Table 4 below, in this system, compared with the base of Sample 3, Samples 1 and 2 were found to have an effect of suppressing apoptosis.
[0015]
Example 2
(1) Test sample Five types of cosmetic raw materials shown in Table 3 below were dissolved in 50% ethanol, 10 μl was added to 1 ml of the medium, and the apoptosis inhibition test of hair follicle-derived epithelial cells according to the present invention was carried out. The amount of fragmented DNA and the total amount of mononucleosomes and oligonucleosomes were measured. The measurement results are shown in Table 4 below.
[0016]
[Table 3]
(2) Evaluation results As shown in Table 4 below, samples 4 to 8 were found to have an effect of suppressing apoptosis.
[0018]
[Table 4]
【The invention's effect】
As described above, the present invention induces hair follicle-derived epithelial cells to undergo apoptosis, and measures the amount of fragmented DNA in the hair follicle-derived epithelial cells and / or the total amount of mononucleosomes and oligonucleosomes. It is clear that a method for evaluating the inhibitory action of apoptosis in more detail is provided, and further an excellent method for evaluating a hair restorer is provided.
Claims (1)
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 JPH1129442A (en) | 1999-02-02 |
| JP3688436B2 true JP3688436B2 (en) | 2005-08-31 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP18252897A Expired - Lifetime JP3688436B2 (en) | 1997-07-08 | 1997-07-08 | Evaluation method of hair restorer |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE329264T1 (en) * | 1998-03-18 | 2006-06-15 | Roche Diagnostics Gmbh | DETECTION OF APOPTOSIS PRODUCTS |
| JP5175437B2 (en) * | 2005-01-28 | 2013-04-03 | ライオン株式会社 | Hair restorer and hair restorer composition |
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1997
- 1997-07-08 JP JP18252897A patent/JP3688436B2/en not_active Expired - Lifetime
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| Publication number | Publication date |
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| JPH1129442A (en) | 1999-02-02 |
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