JP5301146B2 - 特定の方法で培養されたケラチノサイト前駆細胞及びその製造方法 - Google Patents
特定の方法で培養されたケラチノサイト前駆細胞及びその製造方法 Download PDFInfo
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Description
Montagna & Parakkal, The Structure and Function of Skin 172−258 (Academic Press New York, NY, 1974) Cotsarelis, et al. Cell 61, 1329−1337 (1990) Kobayashi, et al. Proc. Natl. Acad. Sci. USA 90, 7391−7395 (1993)
Morris, et al. Nature Biotechnol. 22, 411−417 (2004) Trempus, et al. J. Invest. Dermatol. 120, 501−511 (2003)
Weterings, et al. Brit. J. Dermatol. 104, 1−5 (1981) Limat, et al. Transplantation 59, 1032−1038 (1995)
また、モウズイカの溶媒抽出物を直接体内に導入することも可能である。
ケラチノサイト前駆細胞は、通常培養を継続すると、皮膚における有棘細胞や顆粒細胞のような性質を有するようになる。この細胞では、CD34などの未分化状態を示す分子マーカーの発現が抑制され、フィラグリンやインボルクリンなどの分子マーカーを発現するようになる。
モウズイカの地上部の細断品100gに精製水1kgを加え、95〜100℃、2時間抽出した。得られた抽出液を減圧濃縮し、凍結乾燥してモウズイカ地上部の熱水抽出物を11g得た。
健常人から毛髪を抜去し、毛幹を毛包から約1cm残して取り除いた。毛包を含む毛髪をトリプシン処理してヒト外毛根鞘細胞(hORS)を剥離し、1,000rpm、5分間、室温において遠心分離して回収した。5mlの培地(Humedia KG2; クラボウ)に懸濁し、コラーゲンIVコートした60mm培養皿(ベクトンディッキンソン)に播種した。10日間培養し、細胞を増殖させた後に、4,000個/cm2の密度においてhORSをコラーゲンIVコートした60mm培養皿(ベクトンディッキンソン)に播種し直した。培地中のhORSにモウズイカ抽出物を添加し、CO2インキュベーター中、5%CO2、95%大気、37℃にて2日間培養した。また、コントロールとして、モウズイカ抽出液を添加しない未適用対照を設けた。培養終了時に、細胞よりTORIZOL(Invitrogen社製)にて細胞を溶解することによって総RNAを抽出し、総RNAを基に、RT−PCR法によりmRNA発現量の測定を行った。RT−PCR法にはSuperScriptIII Platinum Two−Step qRT−PCR Kit with SYBR Green(Invitrogen社製)を用い、定量PCR反応に供し、CD34の発現量を定量した。発現量は、GAPDHの発現量にて規格化(ノーマライズ)した。尚、各遺伝子の発現の測定に使用したプライマーは、次の通りである。
TTGCTGCCTTCTGGGTTCAT(配列番号1)
GGTGCAGGCTGGTACTTCCA(配列番号2)
GAPDH用のプライマーセット
TGCACCACCAACTGCTTAGC(配列番号3)
TCTTCTGGGTGGCAGTGATG(配列番号4)
実験例1において調製したhORSを、4,000個/ウェルの密度でコラーゲンIVコートした24ウェルプレートに播種した。モウズイカ(500ng/ml)添加あるいは無添加の条件において3日間培養した。培養後、10%中性ホルマリン溶液(和光)を用いて、4℃、一晩固定した。ラビット抗CD34抗体を反応させた後、蛍光色素Alexa488を連結した抗ウサギIgGを反応させ、蛍光顕微鏡(オリンパス)を用いて観察し、デジタルカメラにて顕微鏡像を撮影した。
実験例1において調製したhORSを60mm培養皿に1x106個播種し、モウズイカ抽出物を添加し、3日間培養を続けた。次に、それぞれの細胞をPBS(−)にて3回洗浄した後、無菌的にラバーポリスマンにて回収し、遠沈後、5%FBS添加ハンクス液(Hank‘s balanced salt solution)に分散し、移植用の細胞として用いた。その後、以下の方法にて、皮膚移植を行い、生着率(%)について測定した。
実験例3において調製したケラチノサイト前駆細胞1x106個を、ヌードマウス(雄性、4週齢)の皮下に移植した。具体的には、移植する1x106個の細胞を、予めCell Tracker(モレキュラープローブ社製)にて蛍光標識し、その時点の蛍光強度を測定し、移植細胞の総数(100%)とした。移植部位をマジックにてマーキングし、移植3日後、7日後に移植部位を摘出し、酵素処理により細胞を分散させ、得られた細胞の蛍光強度を測定し、移植時の蛍光強度(100%)と比較することにより、生着率(%)を算出した。
すなわち、本発明は、再生医療、美容を目的とした外科的施術を伴った治療における、ケラチノサイト前駆細胞の調製方法及び/又は、ケラチノサイト前駆細胞の未分化状態の維持剤として利用が可能である。
Claims (2)
- モウズイカの熱水抽出物を含有することを特徴とするヒトケラチノサイト前駆細胞の未分化状態の維持剤。
- ケラチノサイト前駆細胞がCD34を発現していることを特徴とする請求項1記載の未分化状態の維持剤。
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