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Human iPSCs-based signal disruption reporter assays for predicting developmental toxicants

菅野 聖世 横浜国立大学 DOI:info:doi/10.18880/00014604

2022.05.26

概要

現行の発生毒性試験では、化学物質を妊娠期の母動物に曝露して評価するが、供試動物、時間、コストなどの負担が大きい。そこで発生毒性試験に代わり、生体外で化学物質の発生毒性を評価する手法が提案されてきたが、種差や低スループット性、適用範囲の制限など、それぞれの手法が問題点を抱えている。これらの問題点を解決するため、予測精度の高い生体外発生毒性試験法を開発することが本研究の目的に設定された。本論文は 6 章から構成され、各章の概要は以下の通りであった。

第 1 章 序論
実験動物を用いた出生前発生毒性試験にかかるコストや種差についての問題点、膨大な種類の化学物質が未試験のまま存在する問題点について述べられている。また、これまでに提案されてきた代表的な動物実験代替法をいくつか挙げ、その問題点が示されている。

第 2 章 RTK-SRF シグナルレポーターコンストラクトの作製
本研究で RTK-SRF シグナル伝達経路に注目した根拠となる先行研究が示されている。そして、ゲノム編集用のドナーDNA として用いるプラスミドベクターを作製し、サンガーシーケンスによって塩基置換のないプラスミドベクターを選別した。

第 3 章 ヒトiPS 細胞由来 RTK-SRF シグナルレポーター細胞の樹立
作製したレポーターコンストラクトをドナーDNA として用いてCRISPR-Cas9 システムによってゲノム編集を行い、薬剤選択によって細胞株を樹立した。そして、未分化マーカーの発現とリガンド応答性を評価することで、ヒト iPS 細胞由来 RTK-SRF シグナルレポーター細胞が樹立されたことを確認した。

第 4 章 ECVAM 生体外試験化学物質を用いたシグナル攪乱試験
ECVAM (validation study on in vitro embryotoxicity tests)で使用された化学物質をシグナルレポーター細胞に曝露し、シグナル伝達経路に対する化学物質の影響を検出することで、発生毒性物質を予測することが可能であることが示された。

第 5 章 ICH 試験化学物質を用いたシグナル攪乱試験
Thalidomide を含む ICH 試験化学物質に対して、発生毒性を検出することが可能であることが示された。

第 6 章 結論
第 6 章では、本研究を総括し、今後の展望について述べてある。以上の内容は、博士(工学)として価値あるものとして判断した。

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