JPWO2006073181A1 - 細胞賦活剤、細胞死抑制剤、および細胞死促進剤 - Google Patents
細胞賦活剤、細胞死抑制剤、および細胞死促進剤 Download PDFInfo
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Abstract
Description
発毛、育毛、養毛作用の評価を目的に、各Wnt(Wnt1、3、3a、4、5a、10a、10b、11)を用いて毛乳頭細胞に対する細胞賦活作用を評価した。
WntのcDNAは、一部のWntが市販されているのみであったため、評価に必要なWntの一部は市販品を購入、市販されていないものについては動物組織よりcDNAを調製した。すなわち、Wnt10a、Wnt10b、Wnt11についてはIsogen(ニッポンジーン社製)を用いて、マウスの皮膚組織からmRNAを抽出し、Wntの既知のDNA配列((Genebank:NM 009518;Wnt10a)、(Genebank:NM 011718;Wnt10b)、(Genebank:XM 124961;Wnt11))をもとに、それぞれのプライマー(約20塩基程度)を作製し、PCR法によりそれぞれのcDNAを得た。一方、残りのWnt1、Wnt3、Wnt3a、Wnt4、Wnt5aのcDNAについては、Upstate Biotechnology社より購入(カタログ番号21−121、21−123、21−124、21−125、21−133)した。
Wntタンパク質は活性型での精製が困難であり、そのためWntを安定的に分泌する細胞をまず作製し、その細胞を4日間培養し、培地中にWntが分泌されたもの(conditioned medium:調製培地)をWntの作用評価に用いた。
得られたWnt安定的発現CHO細胞を、10%のFBS(ウシ胎児血清)と、500または750μg/mLのG418を含むHam’sF12培地中で、直径10cmの培養皿に2×104細胞/cm2の密度で播種し、3日間培養した。次いで、培地を無血清CD−CHO培地(Gibco社製)に代え、4日間培養した。培地を回収し、3000rpmで5分間遠心分離し、上澄(調製培地)を得た。一方、親株のCHO細胞を用いて、同様に培養し、遠心分離し、上澄(調製培地)を得、ネガティブコントロール(NC)として調製した。
Wntの毛乳頭細胞の賦活作用を「生細胞数を維持する或いは高める作用」とみなし、細胞内に蓄積される水溶性ホルマザン量を測定することにより推定した。
この細胞賦活作用の作用機序として、無血清条件で惹き起こされる細胞死を抑制するメカニズムが考えられるため、以下のようにしてこのWnt5aの細胞死抑制作用の試験を実施した。
ヒト毛乳頭細胞は、東洋紡から購入し、10%FBS含有DMEM(コージンバイオ社製)を用いて培養した。継代5回目のヒト毛乳頭細胞を、各ウェルあたり0.5×104細胞となるようにI型コラーゲンコート96ウェルマイクロプレート(Becton Dickinson社製)に播種し、24時間培養した。培養液を取り除き血清無添加のDMEM(無血清培地)で2回細胞を洗浄した後、無血清培地、またはリコンビナントWnt5a(R&D社製)を添加した(63〜500ng/ml)無血清培地と交換し、更に24時間、培養を行った。
Wnt5aの細胞からの分泌を促進または抑制する物質をリアルタイムPCRを用いて、Wnt5aの発現量を測定することにより探索した。
オレンジについては、ビターオレンジ(Citrus aurantium L.)の生花を水蒸気蒸留し、精油を採取した残水を、オレンジフラワー水として使用した。
ヒト毛乳頭細胞(hDPC,東洋紡社製)は、10%FBS含有DMEMを用いて維持培養した。
Claims (12)
- Wnt5a及び/又はWnt10aを有効成分とする細胞賦活剤。
- Wnt5a及び/又はWnt10aを有効成分とする細胞死抑制剤。
- Wnt5a及び/又はWnt10aの細胞からの分泌を促進することにより、標的細胞を賦活する細胞賦活剤。
- Wnt5a及び/又はWnt10aの細胞からの分泌を促進することにより、標的細胞の細胞死を抑制する細胞死抑制剤。
- Wnt5a及び/又はWnt10aの細胞からの分泌を抑制することにより、標的細胞の細胞死を促進する細胞死促進剤。
- Wnt5aの細胞からの分泌を促進する物質が、オレンジ、タイム、アルニカ、ホウセンカ、アルギニンから選択される1種または2種以上である、請求項3に記載の細胞賦活剤または請求項4に記載の細胞死抑制剤。
- Wnt5aの細胞からの分泌を抑制する物質が、エキナセアである、請求項5に記載の細胞死促進剤。
- 細胞を賦活、細胞死を抑制または促進する対象の細胞が毛乳頭細胞である、請求項1〜4および請求項6のいずれか1項に記載の細胞賦活剤もしくは細胞死抑制剤、または請求項5および請求項7のいずれか1項に記載の細胞死促進剤。
- 請求項1〜4および請求項6のいずれか1項に記載の細胞賦活剤または細胞死抑制剤を有効成分とする、発毛・育毛・養毛剤。
- 請求項5または請求項7に記載の細胞死促進剤を有効成分とする、脱毛剤。
- Wnt5aまたはWnt5aの細胞からの分泌を促進する物質を有効成分とする、発毛・育毛・養毛剤。
- Wnt10aまたはWnt10aの細胞からの分泌を促進する物質を有効成分とする、発毛・育毛・養毛剤。
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JP2005003190A JP2008179540A (ja) | 2005-01-07 | 2005-01-07 | 細胞分化、発生、増殖を制御する物質および該物質を含む細胞分化・発生・増殖調節剤 |
PCT/JP2006/300090 WO2006073181A1 (ja) | 2005-01-07 | 2006-01-06 | 細胞賦活剤、細胞死抑制剤、および細胞死促進剤 |
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JP2006550906A Pending JPWO2006073181A1 (ja) | 2005-01-07 | 2006-01-06 | 細胞賦活剤、細胞死抑制剤、および細胞死促進剤 |
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JP2008133233A (ja) * | 2006-11-29 | 2008-06-12 | Noevir Co Ltd | Wnt5a産生促進剤、養毛剤 |
JP6186106B2 (ja) * | 2010-10-22 | 2017-08-23 | 株式会社マンダム | 清涼感持続性化粧料 |
KR102418785B1 (ko) * | 2015-09-25 | 2022-07-08 | (주)프로스테믹스 | 식물 착즙물 유래 세포 외 소포체를 포함하는 피부 개선 및 탈모 방지용 조성물 |
JP2017100957A (ja) * | 2015-11-30 | 2017-06-08 | 学校法人東京医科大学 | 永久脱毛剤 |
JP7325075B2 (ja) * | 2018-09-21 | 2023-08-14 | 学校法人 埼玉医科大学 | フリズルド3発現細胞の細胞数増加剤若しくは低下剤、糖尿病予防若しくは治療剤、インスリノーマ予防若しくは治療剤、及びインスリン分泌促進剤若しくは分泌抑制剤よりなる群から選択される少なくとも1つの剤をスクリーニングするためのスクリーニング剤、スクリーニング用キット、及びスクリーニング方法 |
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JP2008179540A (ja) | 2008-08-07 |
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