JPWO2018194124A1 - 体細胞から肺胞上皮細胞への分化用試薬及びその使用 - Google Patents
体細胞から肺胞上皮細胞への分化用試薬及びその使用 Download PDFInfo
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Abstract
Description
[1]NK2ホメオボックスファミリー遺伝子の発現ベクター及びFoxファミリー遺伝子の発現ベクターを含む、体細胞から肺胞上皮細胞への分化用試薬。
[2]NK2ホメオボックスファミリー遺伝子の発現ベクター、Foxファミリー遺伝子の発現ベクター及びGataファミリー遺伝子の発現ベクターを含む、[1]に記載の体細胞から肺胞上皮細胞への分化用試薬。
[3]前記Gataファミリー遺伝子がGata Binding Protein 6(Gata6)遺伝子である、[2]に記載の体細胞から肺胞上皮細胞への分化用試薬。
[4]前記NK2ホメオボックスファミリー遺伝子がNK2 Homeobox 1(Nkx2−1)遺伝子である、[1]〜[3]のいずれかに記載の体細胞から肺胞上皮細胞への分化用試薬。
[5]前記Foxファミリー遺伝子がForkhead Box A1(Foxa1)遺伝子又はForkhead Box A2(Foxa2)遺伝子である、[1]〜[4]のいずれかに記載の体細胞から肺胞上皮細胞への分化用試薬。
[6]体細胞にNK2ホメオボックスファミリー遺伝子及びFoxファミリー遺伝子を強制発現させる工程と、強制発現後の前記体細胞を培養する工程と、を備える、肺胞上皮細胞の製造方法。
[7]NK2ホメオボックスファミリー遺伝子及びFoxファミリー遺伝子を強制発現させる前記工程において、Gataファミリー遺伝子を更に強制発現させる、[6]に記載の製造方法。
[8]前記培養する工程を3次元培養により行う、[6]又は[7]に記載の製造方法。
[9]前記培養する工程を、デキサメタゾン及びFibroblast Growth Factor(FGF)ファミリータンパク質の存在下で行う、[6]〜[8]のいずれかに記載の製造方法。
[10][6]〜[9]のいずれかに記載の製造方法により製造された肺胞上皮細胞。
[11][10]の肺胞上皮細胞を有効成分として含有する細胞医薬。
1実施形態において、本発明は、NK2ホメオボックスファミリー遺伝子の発現ベクター及びFoxファミリー遺伝子の発現ベクターを含む、体細胞から肺胞上皮細胞への分化用試薬を提供する。本実施形態の分化用試薬は、NK2ホメオボックスファミリー遺伝子の発現ベクター及びFoxファミリー遺伝子の発現ベクターを含む組成物であるということもできる。あるいは、NK2ホメオボックスファミリー遺伝子の発現ベクター及びFoxファミリー遺伝子の発現ベクターを含むキットであるということもできる。
1実施形態において、本発明は、体細胞にNK2ホメオボックスファミリー遺伝子及びFoxファミリー遺伝子を強制発現させる工程と、強制発現後の前記体細胞を培養する工程と、を備える、肺胞上皮細胞の製造方法を提供する。
1実施形態において、本発明は、上述した製造方法により製造した肺胞上皮細胞を提供する。本実施形態の肺胞上皮細胞は、例えば、特発性間質性肺炎、インフルエンザや細菌による肺炎、急性呼吸窮迫症候群(ARDS)、慢性閉塞性肺疾患(COPD)等の肺胞上皮細胞の移植が有効な疾患の治療等に用いることができる。
1実施形態において、本発明は、上述した製造方法により製造された肺胞上皮細胞を有効成分として含有する細胞医薬を提供する。本実施形態の細胞医薬は、例えば、特発性間質性肺炎、インフルエンザや細菌による肺炎、急性呼吸窮迫症候群(ARDS)、慢性閉塞性肺疾患(COPD)等の肺胞上皮細胞の移植が有効な疾患の治療等に用いることができる。本実施形態の細胞医薬において、肺胞上皮細胞は、患者自身の体細胞から製造されたものであってもよいし、主要組織適合遺伝子複合体が適合する第3者の体細胞から製造されたものであってもよい。
1実施形態において、本発明は、上述した製造方法により製造された肺胞上皮細胞の有効量を、治療を必要とする患者に移植することを含む肺疾患の治療方法を提供する。肺疾患としては、例えば、特発性間質性肺炎、インフルエンザや細菌による肺炎、急性呼吸窮迫症候群(ARDS)、慢性閉塞性肺疾患(COPD)等が挙げられる。
(体細胞から肺胞上皮細胞への分化に必要な因子の検討1)
体細胞から肺胞上皮細胞への分化に必要な因子を検討した。体細胞として、マウス胎仔由来の線維芽細胞を使用した。まず、マウス線維芽細胞に、レトロウイルス発現ベクターを用いて、下記表1に示す14種類のマウス遺伝子を同時に導入して過剰発現させた。表1には、各遺伝子のNCBI Gene ID及びMGI IDも記載した。
(体細胞から肺胞上皮細胞への分化に必要な因子の検討2)
体細胞から肺胞上皮細胞への分化に必要な因子のより詳細な検討を行った。具体的には、まず、表1に示す14種類の遺伝子から1種類ずつ遺伝子を除いた13種類の遺伝子をマウス線維維芽細胞に導入した以外は実験例1と同様にして、肺胞上皮細胞のマーカーであるSftpc遺伝子の発現量を定量的RT−PCRにより測定した。また、対照として、表1に示す遺伝子の代わりにGFPのレトロウイルス発現ベクターを導入した線維芽細胞を用いた。
(体細胞から肺胞上皮細胞への分化に必要な因子の検討3)
実験例2の結果から、体細胞から肺胞上皮細胞への分化にNkx2−1遺伝子の発現が必須であることが明らかとなった。そこで、Nkx2−1遺伝子と、表1に示すNkx2−1遺伝子以外の13種類の遺伝子から選択した1種類の遺伝子との組み合わせをマウス線維芽細胞に導入し、肺胞上皮細胞のマーカーであるSftpc遺伝子及びScgb1a1遺伝子の発現量を定量的RT−PCRにより測定した。また、対照として、表1に示す遺伝子の代わりにGFPのレトロウイルス発現ベクターを導入した線維芽細胞を用いた。
(体細胞から肺胞上皮細胞への分化に必要な因子の検討4)
実験例2、3の結果に基づいて、体細胞から肺胞上皮細胞への分化に必要な因子の検討を更に行った。具体的には、表1に示す番号1の因子(Nkx2−1遺伝子)、番号2の因子(Foxa1遺伝子)、番号3の因子(Foxa3遺伝子)及び番号8の因子(Gata6遺伝子)を様々な組み合わせでマウス線維芽細胞に導入し、肺胞上皮細胞のマーカーであるSftpc遺伝子及びScgb1a1遺伝子の発現量を定量的RT−PCRにより測定した。また、対照として、表1に示す遺伝子の代わりにGFPのレトロウイルス発現ベクターを導入した線維芽細胞を用いた。
(体細胞から肺胞上皮細胞への分化条件の検討1)
SPC−GFPマウスは、SPC(Sftpc)遺伝子のプロモーターの下流にGFP遺伝子が連結されたコンストラクトが導入されたトランスジェニックマウスである。SPC−GFPマウスでは、Sftpc遺伝子を発現する細胞でGFPの蛍光が観察される。そこで、体細胞として、SPC−GFPマウスの胎仔由来の線維芽細胞を使用して肺胞上皮細胞への分化条件の検討を行った。
(体細胞から肺胞上皮細胞への分化条件の検討2)
SPC−GFPマウスの線維芽細胞に、表1に示す番号1の因子(Nkx2−1遺伝子)、番号2の因子(Foxa1遺伝子)、番号3の因子(Foxa3遺伝子)及び番号8の因子(Gata6遺伝子)の合計4種類の遺伝子を導入して1〜2日間2次元培養した。
(体細胞から肺胞上皮細胞への分化条件の検討3)
SPC−GFPマウスの線維芽細胞に、表1に示す番号1の因子(Nkx2−1遺伝子)、番号2の因子(Foxa1遺伝子)及び番号8の因子(Gata6遺伝子)の合計3種類の遺伝子、又は番号1の因子(Nkx2−1遺伝子)、番号3の因子(Foxa2遺伝子)及び番号8の因子(Gata6遺伝子)の合計3種類の遺伝子をそれぞれ導入し、実験例6と同様に3次元培養した。
(分化誘導した肺胞上皮細胞のインフルエンザマウスへの移植)
マウス馴化インフルエンザウイルス(A/H1N1、PR8)を、200p.f.u./マウスの投与量で点鼻感染させた。また、対照として、インフルエンザウイルスの代わりにリン酸緩衝生理食塩水(PBS)を投与したマウスを用意した。
続いて、各群のマウスの体重及び生存率の推移を観察した。図8(a)は、各群のマウスの体重の推移を示すグラフであり、図8(b)は、各群のマウスの生存率の推移を示すグラフである。図8(a)及び(b)中、「Flu+iPUL」は、マウスにインフルエンザウイルスを感染させ、胚性線維芽細胞から分化誘導した肺胞上皮細胞(iPUL)を投与した群の結果であることを示し、「Flu+MEF」は、マウスにインフルエンザウイルスを感染させ、分化誘導を行っていない胚性線維芽細胞を投与した群の結果であることを示し、「Flu+PBS」は、マウスにインフルエンザウイルスを感染させ、PBSを投与した群の結果であることを示し、「PBS+iPUL」は、マウスにPBSを投与し、続いて胚性線維芽細胞から分化誘導した肺胞上皮細胞を投与した群の結果であることを示し、「PBS+MEF」は、マウスにPBSを投与し、続いて分化誘導を行っていない胚性線維芽細胞を投与した群の結果であることを示す。
マウスにインフルエンザウイルスを感染させ、胚性線維芽細胞から分化誘導した肺胞上皮細胞を投与した群(Flu+iPUL群)、及び、マウスにインフルエンザウイルスを感染させ、分化誘導を行っていない胚性線維芽細胞を投与した群(Flu+MEF群)の各群のマウスについて、インフルエンザウイルスの感染から5日目にそれぞれ肺を摘出した。続いて、摘出した肺を4%パラホルムアルデヒドにて固定し、パラフィン包埋組織切片を作製した。続いて、各群の組織切片を、エタノール及び純水にて脱パラフィン処理を行い、ヘマトキシリン・エオジン染色し、光学顕微鏡で観察した。
続いて、Flu+iPUL群の肺の組織切片を免疫染色し、GFPタンパク質及びSP−Cタンパク質の発現を検討した。
(a)及び(b)はそれぞれ視野及び倍率が異なっている。図10(a)及び(b)中、GFPタンパク質及びSP−Cタンパク質の二重陽性細胞を矢印で示す。
(ヒト線維芽細胞から肺胞上皮細胞への分化誘導)
ヒト成人皮膚由来線維芽細胞(細胞名「HDFa」、サーモフィッシャーサイエンティフィック社)に、ヒトNKX2−1遺伝子、ヒトFOXA1遺伝子、ヒトFOXA2遺伝子、ヒトGATA6遺伝子を組み合わせて導入し、マウスと同様に肺胞上皮細胞に分化誘導できるか否かを検討した。
Claims (11)
- NK2ホメオボックスファミリー遺伝子の発現ベクター及びFoxファミリー遺伝子の発現ベクターを含む、体細胞から肺胞上皮細胞への分化用試薬。
- Gataファミリー遺伝子の発現ベクターを更に含む、請求項1に記載の体細胞から肺胞上皮細胞への分化用試薬。
- 前記Gataファミリー遺伝子がGata Binding Protein 6(Gata6)遺伝子である、請求項2に記載の体細胞から肺胞上皮細胞への分化用試薬。
- 前記NK2ホメオボックスファミリー遺伝子がNK2 Homeobox 1(Nkx2−1)遺伝子である、請求項1〜3のいずれか一項に記載の体細胞から肺胞上皮細胞への分化用試薬。
- 前記Foxファミリー遺伝子がForkhead Box A1(Foxa1)遺伝子又はForkhead Box A2(Foxa2)遺伝子である、請求項1〜4のいずれか一項に記載の体細胞から肺胞上皮細胞への分化用試薬。
- 体細胞にNK2ホメオボックスファミリー遺伝子及びFoxファミリー遺伝子を強制発現させることと、
強制発現後の前記体細胞を培養することと、
を備える、肺胞上皮細胞の製造方法。 - 前記NK2ホメオボックスファミリー遺伝子及びFoxファミリー遺伝子を強制発現させることにおいて、Gataファミリー遺伝子を更に強制発現させる、請求項6に記載の製造方法。
- 前記培養する工程を3次元培養により行う、請求項6又は7に記載の製造方法。
- 前記培養する工程を、デキサメタゾン及びFibroblast Growth Factor(FGF)ファミリータンパク質の存在下で行う、請求項6〜8のいずれか一項に記載の製造方法。
- 請求項6〜9のいずれか一項に記載の製造方法により製造された肺胞上皮細胞。
- 請求項10の肺胞上皮細胞を有効成分として含有する細胞医薬。
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