JPH07274954A - Culture of hair matrix cells - Google Patents
Culture of hair matrix cellsInfo
- Publication number
- JPH07274954A JPH07274954A JP6072990A JP7299094A JPH07274954A JP H07274954 A JPH07274954 A JP H07274954A JP 6072990 A JP6072990 A JP 6072990A JP 7299094 A JP7299094 A JP 7299094A JP H07274954 A JPH07274954 A JP H07274954A
- Authority
- JP
- Japan
- Prior art keywords
- hair
- cells
- culture
- vascular endothelial
- culturing
- 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.)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、毛母細胞培養法、詳し
くは生理的な状態の増殖・分化能力を有した毛母細胞を
定常的に培養し得る毛母細胞培養法、及びこれを利用し
た被験物質の育毛作用の有無のスクリーニング方法に関
する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hair mother cell culturing method, and more specifically to a hair mother cell culturing method capable of steadily culturing hair mother cells having a physiological state of proliferation / differentiation ability, and The present invention relates to a screening method for the presence or absence of a hair-growing action of a test substance used.
【０００２】[0002]
【従来の技術】毛母細胞は毛の成長を支える基となる細
胞であることから、男性型脱毛症いわゆる若禿をはじめ
多くの脱毛症の改善を目的として、毛母細胞増殖促進因
子の探索が広く行われている。上記因子を効率的及び効
果的に探索するには、生体応答を簡便に、より正確に調
べることが必要とされ、従来から毛母細胞を単離培養す
る種々の方法が試みられている。2. Description of the Related Art Since hair mother cells are the base cells that support hair growth, a search for hair mother cell growth-promoting factors has been carried out for the purpose of improving many alopecia including androgenetic alopecia, so-called baldness. Is widely practiced. In order to search for the above factors efficiently and effectively, it is necessary to examine biological responses simply and more accurately, and various methods for isolating and culturing hair mother cells have been attempted.
【０００３】このような単離培養法は大別して、細胞培
養法と器官培養法とに分類される。細胞培養法の一例と
して、適当な方法によって単離した毛母細胞を適当な塩
類溶液（培養液）中で培養する方法が報告されている
が、この培養法では毛母細胞の成育が悪いため、牛胎児
血清又は脳下垂体抽出物等を添加して培養を行う方法
（特開昭６３−３０１７８５号公報）や、グルコサミノ
グリカン又は線維芽細胞増殖因子を添加して培養を行う
方法（特開平４−１８７６２２号公報）が提案されてい
る。一方、毛母細胞を含有する器官である毛包組織を培
養する器官培養法としては、例えば、Ｂｕｈｌ．Ａ．Ｅ
等の方法（Ｊ．Ｉｎｖｅｓｔ．Ｄｅｒｍａｔｏｌ，９
２，３１５，１９８９）などが知られている。Such isolation culture methods are roughly classified into cell culture methods and organ culture methods. As an example of a cell culture method, a method of culturing hair mother cells isolated by an appropriate method in an appropriate salt solution (culture solution) has been reported. However, this growth method results in poor growth of hair mother cells. , A method of culturing by adding fetal bovine serum or a pituitary extract (JP-A-63-301785), and a method of culturing by adding glucosaminoglycan or fibroblast growth factor ( JP-A-4-187622) has been proposed. On the other hand, as an organ culture method for culturing hair follicle tissue, which is an organ containing hair mother cells, for example, Buhl. A. E
Et al. (J. Invest. Dermatol, 9
2,315,1989) and the like are known.
【０００４】[0004]
【発明が解決しようとする課題】しかしながら、上記器
官培養法は、得られる細胞の数が少ない、固体間の差が
大きい、長期的に培養することができないなどの問題点
があった。また、これまでに提案されている毛母細胞の
単離培養法は、一定条件下で定常的に毛母細胞を培養し
得るが、毛母細胞の増殖力は低く、毛の成長を生理的に
正しく反映したものとはいい難い。However, the above-mentioned organ culture method has problems that the number of cells to be obtained is small, the difference between the solids is large, and the cells cannot be cultured for a long period of time. In addition, the hair mother cell isolation culture methods that have been proposed so far can steadily culture hair mother cells under certain conditions, but the proliferative ability of hair mother cells is low, and hair growth is physiologically performed. It is hard to say that it was correctly reflected in.
【０００５】従って、本発明の目的は、生理的な状態の
増殖・分化能力を有する毛母細胞を定常的に培養し得る
毛母細胞培養法、及びこれを利用した被験物質の育毛作
用の有無のスクリーニング方法を提供することにある。Therefore, the object of the present invention is to provide a hair mother cell culturing method capable of steadily culturing hair mother cells having the ability to proliferate / differentiate in a physiological state, and the presence or absence of a hair-growing action of a test substance using the same. The present invention is to provide a screening method.
【０００６】[0006]
【課題を解決するための手段】このような実情におい
て、本発明者は鋭意検討を行った結果、毛母細胞を培養
する際に、血管内皮細胞、血管内皮細胞膜画分又は血管
内皮細胞培養上清を共存させた場合、生理的な状態の増
殖・分化能力を有する毛母細胞を定常的に培養すること
ができ、また、毛母細胞と血管内皮細胞を混合すること
により被験物質の存在下において毛母細胞の増殖促進率
を調節し得るので、毛母細胞の培養を当該成分及び被験
物質の存在下で行った場合、被験物質のスクリーニング
を確実に行うことができることを見出し、本発明を完成
した。Under such circumstances, the present inventor has conducted diligent studies, and as a result, when culturing hair mother cells, vascular endothelial cells, vascular endothelial cell membrane fraction or vascular endothelial cell culture When coexisting with Qing, hair mother cells capable of proliferating / differentiating in a physiological state can be constantly cultured, and by mixing hair mother cells and vascular endothelial cells in the presence of a test substance. Since it is possible to regulate the proliferation promoting rate of hair mother cells in, in the case of culturing hair mother cells in the presence of the component and the test substance, it was found that the test substance can be reliably screened, the present invention completed.
【０００７】即ち、本発明は、毛母細胞を、血管内皮細
胞、血管内皮細胞膜画分又は血管内皮細胞培養上清の存
在下に培養することを特徴とする毛母細胞培養法を提供
するものである。また、本発明は、上記毛母細胞の培養
を被験物質の存在下で行うことを特徴とする被験物質の
育毛作用の有無のスクリーニング方法を提供するもので
ある。That is, the present invention provides a method for culturing hair matrix cells, which comprises culturing hair matrix cells in the presence of vascular endothelial cells, vascular endothelial cell membrane fraction or vascular endothelial cell culture supernatant. Is. The present invention also provides a method for screening the presence or absence of hair-growth action of a test substance, which comprises culturing the above hair mother cells in the presence of the test substance.
【０００８】本発明の培養対象である毛母細胞として
は、ヒトに限らず毛髪を有する動物種の毛母細胞であれ
ばよく、その部位、雌雄、齢、毛髪の発育状態を問わな
いが、望ましくは齧歯類動物の新生児体毛から採取した
毛母細胞を用いる。材料からの毛母細胞調製法として
は、外科的手法（Ｊｏｎｅｓ，Ｌ．，Ｎ．，ｅｔ ａ
ｌ，Ｊ Ｉｎｖｅｓｔ Ｄｅｒｍａｔｏｌ，９０，５
８，１９８８）、酵素処理を用いた手法（Ｒｏｇｅｒ
ｓ，Ｇ．，ｅｔ ａｌ，Ｊ ＩｎｖｅｓｔＤｅｒｍａｔ
ｏｌ，８９，３６９，１９８７；Ｍａｒｔｉｎ，Ｒ．，
Ｇ．，ｅｔａｌ，Ｊ Ｉｎｖｅｓｔ Ｄｅｒｍａｔｏ
ｌ，８７，７６８，１９８６）等が挙げられるが、本発
明においてはいずれの手法をも用いることができる。The hair mother cells to be cultured in the present invention are not limited to humans, and may be hair mother cells of animal species having hair, regardless of the site, sex, age and hair growth state. Desirably, hair matrix cells collected from neonatal body hair of rodents are used. As a method for preparing hair matrix cells from the material, a surgical method (Jones, L., N., et a.
1, J Invest Dermatol, 90 , 5
8, 1988), a method using enzyme treatment (Roger
S.G. , Et al, J Invest Dermat
Ol, 89 , 369, 1987; Martin, R .; ，
G. , Et al, J Invest Dermato
1, 87 , 768, 1986) and the like, but any method can be used in the present invention.
【０００９】一方、本発明で用いる血管内皮細胞の材料
としては、毛母細胞の場合と同様のものから採取したも
のを用いることができるが、両組織の由来を同一にする
必要はなく、毛母細胞とは異なる種、部位であっても問
題はない。血管内皮細胞の調製法としては、外科的手法
（Ｊａｆｆｅ，Ｅ．，Ａ．，Ｊ．Ｃｌｉｎ．Ｉｎｖｅｓ
ｔ．，５２，２７４５，１９７３）、酵素処理と密度勾
配遠心法を用いた手法（Ｂｏｗｍａｎ，Ｐ．，Ｄ．，Ｂ
ｅｔｚ，Ａ．，Ｌ．，Ａｒ，Ｄ．，ｅｔ ａｌ，Ｉｎ
Ｖｉｔｒｏ．１７，３５３，１９８１）等があるが、市
販されている血管内皮細胞を用いることもできる。On the other hand, as the material for the vascular endothelial cells used in the present invention, those obtained from the same material as that of the hair mother cells can be used, but it is not necessary to make the origins of both tissues the same. There is no problem even if the species and site are different from the mother cell. As a method for preparing vascular endothelial cells, a surgical method (Jaffe, E., A., J. Clin. Invests) is used.
t. , 52 , 2745, 1973), a method using enzyme treatment and density gradient centrifugation (Bowman, P., D., B).
etz, A .; L. Ar, D .; , Et al, In
Vitro. 17 , 353, 1981) and the like, but commercially available vascular endothelial cells can also be used.
【００１０】また、本発明においては、上記血管内皮細
胞から一般的に用いられている細胞膜画分調製法により
得られた血管内皮細胞膜画分、及び血管内皮細胞を培養
して得られた培養上清及びその分画物を上記血管内皮細
胞と同様に用いることができる。Further, in the present invention, the vascular endothelial cell membrane fraction obtained from the above-mentioned vascular endothelial cells by a generally used cell membrane fraction preparation method, and the culture obtained by culturing the vascular endothelial cells The supernatant and its fraction can be used in the same manner as the above-mentioned vascular endothelial cells.
【００１１】本発明を実施するには、上記毛母細胞と上
記血管内皮細胞、上記血管内皮細胞膜画分又は上記血管
内皮細胞培養上清とを、細胞数、蛋白質量又はＤＮＡ量
の比が好ましくは１：９９〜９９：１の範囲で、特に好
ましくは１：９〜９：１の範囲で混合して培養する。培
養はいずれか一方の細胞をあらかじめ培養し、他方を後
から加えてもよく、また、あらかじめ両者を混合してお
き、同時に培養してもよい。In practicing the present invention, the hair matrix cells and the vascular endothelial cells, the vascular endothelial cell membrane fraction or the vascular endothelial cell culture supernatant are preferably in a ratio of cell number, protein amount or DNA amount. Is mixed in the range of 1:99 to 99: 1, particularly preferably in the range of 1: 9 to 9: 1, and cultured. For culturing, either one of the cells may be cultivated in advance and the other may be added later, or both may be mixed in advance and cultivated at the same time.
【００１２】培養方法としては浮遊培養法、付着培養
法、包埋培養法などのいずれの方法も用いることができ
るが、コラーゲン、寒天などの支持体を用いた包埋培養
法が好ましい。また、その培養条件としては、２０〜３
８℃、０〜１０％ＣＯ2 、及び０〜９５％Ｏ2 含有気相
下とすることが好ましい。As the culturing method, any of floating culture method, adherent culture method, embedded culture method and the like can be used, but the embedded culture method using a support such as collagen or agar is preferable. The culture conditions are 20 to 3
It is preferable to use a gas phase containing 8 ° C., 0 to 10% CO 2 , and 0 to 95% O 2 .
【００１３】本発明の毛母細胞培養法を用いて被験物質
の育毛作用の有無のスクリーニングをするには、毛母細
胞を、血管内皮細胞、血管内皮細胞膜画分及び血管内皮
細胞培養上清から選ばれる血管内皮細胞群並びに被験物
質の存在下に培養して、被験物質の添加による毛母細胞
の増殖増進作用を検討することにより行われる。In order to screen the presence or absence of a hair-growing effect of a test substance using the hair mother cell culture method of the present invention, hair mother cells are isolated from vascular endothelial cells, vascular endothelial cell membrane fractions and vascular endothelial cell culture supernatants. It is carried out by culturing in the presence of a selected vascular endothelial cell group and a test substance, and examining the effect of addition of the test substance on the promotion of proliferation of hair mother cells.
【００１４】[0014]
【発明の効果】本発明の毛母細胞培養法によれば、生理
的な状態の増殖・分化能力を有した毛母細胞が定常的に
培養されるので、育毛剤の開発研究や毛包組織細胞間相
互作用など多方面にわたる毛の基礎研究に利用すること
ができ、また、毛の発生に関与し得る因子をとらえるこ
とができるので、永年未解決であった脱毛機構を解明す
る手段として有用である。EFFECTS OF THE INVENTION According to the hair mother cell culture method of the present invention, hair mother cells having a physiologically proliferative / differentiating ability are constantly cultured. It can be used for basic research of hair in various fields such as cell-cell interaction, and since it can grasp factors that may be involved in hair development, it is useful as a means to elucidate the hair loss mechanism that has been unsolved for many years. Is.
【００１５】[0015]
【実施例】以下、実施例を挙げて本発明をさらに詳細に
説明するが、本発明はこれらの実施例に限定されるもの
ではない。 実施例１ （毛母細胞を得る方法） 新生マウスを氷冷麻酔し、７０％エタノールで皮膚を消
毒した。次に皮膚を採取し、皮下組織等を取り除いた
後、約３ｍｍ幅の短冊状に裁断した。この皮膚をリン酸
緩衝生理食塩水（ＰＢＳ）で洗浄した後、０．１２５％
のトリプシン溶液を用い、４℃下で一昼夜消化した。ト
リプシン処理後、ピンセットで丁寧に表皮を剥離除去し
て真皮組織を得た。得られた真皮をＰＢＳで洗浄した
後、３７℃下で０．２５％コラゲナーゼ溶液で攪拌しな
がら処理した。さらにＤＮａｓｅ Ｉ（５０００単位）
加え、ナイロン網（１５０Ｍ／Ｓ）に瀘過することで残
存組織片を除去した組織懸濁液を液体培地（Ｍｅｄｉｕ
ｍ１９９）で洗浄した。この組織懸濁液を１０％（Ｗ／
Ｖ）Ｆｉｃｏｌｌ４００で密度勾配による遠心分離を行
った。これにより、下層（沈殿物）に毛母細胞が得られ
る。得られた沈殿物に対してさらに上記密度勾配遠心分
離を繰り返し、遠心処理（５０Ｇ、４分）で２回洗浄
し、毛母細胞懸濁液を得た。The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Example 1 (Method for obtaining hair matrix cells) Newborn mice were anesthetized with ice and the skin was disinfected with 70% ethanol. Next, the skin was collected, and after removing the subcutaneous tissue and the like, it was cut into strips with a width of about 3 mm. After washing the skin with phosphate buffered saline (PBS), 0.125%
Digestion was performed overnight at 4 ° C. using the trypsin solution of. After the trypsin treatment, the epidermis was carefully peeled off with tweezers to obtain a dermal tissue. The obtained dermis was washed with PBS and then treated with a 0.25% collagenase solution at 37 ° C. with stirring. Furthermore, DNase I (5000 units)
In addition, the tissue suspension from which residual tissue pieces were removed by filtering through a nylon mesh (150 M / S) was used as a liquid medium (Media).
m199). This tissue suspension is 10% (W /
V) Centrifugation was performed on a Ficoll 400 with a density gradient. As a result, hair matrix cells are obtained in the lower layer (precipitate). The density gradient centrifugation was further repeated on the obtained precipitate, and the precipitate was washed twice by centrifugation (50 G, 4 minutes) to obtain a hair matrix suspension.
【００１６】実施例２ （毛母・血管内皮細胞混合培養
法） コラーゲン・ゲル溶液（Ｍｅｄｉｕｍ１９９で平衡化し
たもの）と毛母細胞懸濁液（５×１０5ｃｅｌｌｓ／ｍ
ｌ）と血管内皮細胞懸濁液（２．５×１０4ｃｅｌｌｓ
／ｍｌ）とを体積比２：２：１で混合して攪拌し、これ
をマイクロ・ウエル・プレートに植え込み、ゲルの硬化
が観察された後、ゲルと同量のＭｅｄｉｕｍ１９９を添
加した。これを５％ＣＯ2 インキュベーター内（３７
℃）で２日間培養し、3Ｈ−チミジン取り込み等による
細胞増殖試験を行ったところ、表１に示すように、毛母
細胞単独培養時に比べて混合培養時のそれが著しく高揚
していた。Example 2 (Mixed Culture Method of Hair Mother / Vascular Endothelial Cells) Collagen / gel solution (equilibrated with Medium 199) and hair mother cell suspension (5 × 10 5 cells / m)
l) and vascular endothelial cell suspension (2.5 × 10 4 cells)
/ Ml) was mixed in a volume ratio of 2: 2: 1 and stirred, and this was seeded in a micro well plate, and after hardening of the gel was observed, the same amount of Medium 199 as the gel was added. This is put in a 5% CO 2 incubator (37
When cultured for 2 days at (° C.) and subjected to a cell proliferation test by incorporation of 3 H-thymidine, etc., as shown in Table 1, it was remarkably enhanced in the mixed culture as compared with the single culture of hair mother cells.
【００１７】[0017]
【表１】 [Table 1]
【００１８】実施例３ （混合培養法における発毛促進
剤の効果） 毛母細胞と血管内皮細胞との混合培養法において、種々
の濃度で発毛促進剤（オトギリ草エキス：特開平３−１
６３０２６号公報参照）を添加したときの細胞増殖能を
測定した。結果を表２に示す。表２から、毛母細胞と血
管内皮細胞との混合培養法は、毛母細胞単独培養法や毛
母細胞と毛乳頭細胞との混合培養法と比較して、極めて
効果的に被験物質の発毛促進活性の有無を検出し得るこ
とが分かる。Example 3 (Effect of hair growth-promoting agent in mixed culture method) In a mixed culture method of hair mother cells and vascular endothelial cells, a hair growth promoter (Hypericum perforatum extract: Japanese Patent Application Laid-Open No. 3-1) at various concentrations.
(See Japanese Patent Laid-Open No. 63026), the cell proliferation ability was measured. The results are shown in Table 2. From Table 2, the mixed culture method of hair mother cells and vascular endothelial cells was much more effective than the cultured culture method of hair mother cells alone or the mixed culture method of hair mother cells and hair papilla cells. It can be seen that the presence or absence of hair promoting activity can be detected.
【００１９】[0019]
【表２】 [Table 2]
Claims (2)
胞膜画分又は血管内皮細胞培養上清の存在下に培養する
ことを特徴とする毛母細胞培養法。1. A method for culturing hair matrix cells, which comprises culturing hair matrix cells in the presence of vascular endothelial cells, a vascular endothelial cell membrane fraction, or a vascular endothelial cell culture supernatant.
で行うことを特徴とする被験物質の育毛作用の有無のス
クリーニング方法。2. A method of screening for the presence or absence of a hair-growing action of a test substance, which comprises culturing the hair matrix cells in the presence of the test substance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6072990A JPH07274954A (en) | 1994-04-12 | 1994-04-12 | Culture of hair matrix cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6072990A JPH07274954A (en) | 1994-04-12 | 1994-04-12 | Culture of hair matrix cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07274954A true JPH07274954A (en) | 1995-10-24 |
Family
ID=13505359
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6072990A Pending JPH07274954A (en) | 1994-04-12 | 1994-04-12 | Culture of hair matrix cells |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07274954A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10265341A (en) * | 1997-03-26 | 1998-10-06 | Shiseido Co Ltd | Assay of hair-glowing agent |
| JP2018082638A (en) * | 2016-11-21 | 2018-05-31 | 国立大学法人横浜国立大学 | Method for producing assembly of regenerative hair follicle germ having microvascular structure, hair follicle tissue-containing sheet, and method for producing hair follicle tissue-containing sheet |
| JP2018528784A (en) * | 2015-09-29 | 2018-10-04 | ロレアル | Use of hair matrix cells to prepare microfollicles |
-
1994
- 1994-04-12 JP JP6072990A patent/JPH07274954A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10265341A (en) * | 1997-03-26 | 1998-10-06 | Shiseido Co Ltd | Assay of hair-glowing agent |
| JP2018528784A (en) * | 2015-09-29 | 2018-10-04 | ロレアル | Use of hair matrix cells to prepare microfollicles |
| JP2018082638A (en) * | 2016-11-21 | 2018-05-31 | 国立大学法人横浜国立大学 | Method for producing assembly of regenerative hair follicle germ having microvascular structure, hair follicle tissue-containing sheet, and method for producing hair follicle tissue-containing sheet |
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