骨髄幹細胞の可塑性とそのメカニズム

• Project/Area Number: 14081202
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: The University of Tokyo
• Principal Investigator: 中内 啓光 東京大学, 医科学研究所, 教授 (40175485)
• Co-Investigator(Kenkyū-buntansha): 依馬 秀夫 東京大学, 医科学研究所, 産学連携研究員(特任助教) (50344445) ; 江藤 浩之 東京大学, 医科学研究所, 助手 (50286986) ; 岩間 厚志 千葉大学, 大学院・医学系研究院, 教授 (70244126)
• Project Period (FY): 2002 – 2006
• Project Status: Completed (Fiscal Year 2006)
• Budget Amount: ¥121,500,000 (Direct Cost: ¥121,500,000); Fiscal Year 2006: ¥24,900,000 (Direct Cost: ¥24,900,000); Fiscal Year 2005: ¥24,900,000 (Direct Cost: ¥24,900,000); Fiscal Year 2004: ¥24,900,000 (Direct Cost: ¥24,900,000); Fiscal Year 2003: ¥23,400,000 (Direct Cost: ¥23,400,000); Fiscal Year 2002: ¥23,400,000 (Direct Cost: ¥23,400,000)
• Keywords: 造血幹細胞 / 自己複製 / bcrp-1 / Lnk / サイトカイン / Endomucin / STAT5 / TMTSP / myeloproliferative disorder / ポリコーム群タンパク / Bmi-1 / 精子幹細胞 / 自己複製能 / Repopulation Unit / Competitive Repopulation Unit / ポリコーム遺伝子群 / 非対称性分裂 / 対娘細胞解析 / blastcyst / 可塑性 / SP細胞 / 活性型STAT

Research Abstract

1)造血幹細胞の多様性と階層性の解明
5あるいは6カラー解析とソーティングを用いることにより高度に純化された造血幹細胞分画であるCD34-KSL細胞をさらに他の抗原で細分画することを試みた。50以上の抗体をスクリーニングしたが、CD34-KSL分画をさらに一部のみ染色する抗体は僅かで、ほとんどは全て陽性あるいは陰性であった。その中でSLAMファミリーの一つで造血幹細胞のマーカーとして最近報告されたCD150はCD34-KSL細胞をある程度分画したのでCD150強陽性CD34-KSL細胞とCD150陰性CD34-KSL細胞について移植実験を行った。その結果、どちらの分画も同じ頻度で長期骨髄再建能を持つ細胞を含んでいたが、CD150強陽性分画の細胞の方が立ち上がりが遅い傾向が認められ、CD150の発現と造血幹細胞の未分化性に相関があることが示唆された。
同様にSP分画細胞とCD34-KSL細胞を6カラー解析によって詳細に解析を行ったところCD34-KSL細胞の半分はSP分画にあるが、半分は非SP分画にあること、そしてどちらの分画にも同じ数の造血幹細胞が含まれていて活性も同等であった。Bcrp-1の発現は遺伝子レベル、タンパクレベルでも差はなかった。さらにSP-CD34-KSL細胞を移植しても非SP-CD34-KSL細胞が出現するし、その逆も起こりえることが示された。また、bcrp-1を造血幹細胞に遺伝子導入して強制発現させるとSP細胞は増加したが、そこに含まれる造血幹細胞の数は増加していなかった。以上より、bcrp-1の発現が造血幹細胞活性とかなり強く相関しているものの、造血幹細胞の自己複製や分化とbcrp-1の機能の間には直接的な関連性は低いことが示唆された。
2)造血幹細胞の多分化能および自己複製能を規定する分子の同定
アダプター分子であるLnkが自己複製を負に制御すること、Lnk欠損造血幹細胞が野生型造血幹細胞に比してTPOに対して高感受性であり、TPOの刺激によって有意に対称性自己複製の頻度が高くなることを実験的に示した。これらのデータはLnkがTPOの受容体であるc-mplの下流にある自己複製シグナルを負に制御する分子であることを示している。そこでc-mpl下流のどのシグナル伝達経路が関与しているかを明らかにするため、我々が開発したin-droplet single cell immunostaining法を基本にしたSingle Cell Immunophophorylation AssayによりTPO刺激後の各種シグナル伝達系のリン酸化状況を解析した。その結果、Lnk欠損造血幹細胞においてはSTAT5ならびにAktのリン酸化が野生型造血幹細胞と比較して著明に亢進していること、P38MAPKの不活性化が亢進していることが示され、造血幹細胞の自己複製に関与するシグナル伝達系を始めて明らかにすることができた。

Research Products

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  • Author(s): 八代嘉美, 中内啓光
  • Total Pages: 166
  • Publisher: 日本実業出版社
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