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遺伝子導入マウスヘパトーマ細胞を用いた中空糸型バイオ人工肝臓装置の設計および評価

藤井, 祐輔 FUJII, Yusuke フジイ, ユウスケ 九州大学

2021.03.24

概要

肝臓は生体における代謝の中心臓器であり、重篤な不全状態に陥った場合、肝移植しか有効な治療法が存在しない。一方、肝移植はドナー不足が深刻な問題であり、肝移植に代わる、あるいは肝移植までの橋渡しとしての代替医療の確立が望まれている。バイオ人工肝臓は、培養肝細胞を用いて患者の体外から強力な肝機能補助を行う治療概念であり、肝移植の代替治療として盛んに研究されてきた。一方、バイオ人工肝臓の実用化には臨床用細胞源の確保という大きな課題がある。高機能発現が可能で、容易に大量供給が可能な細胞源として、動物肝細胞、肝細胞株、幹細胞など、多くの候補細胞が検討されてきたが、種々の要求を十分に満たす決定的な細胞源は未だ見いだせておらず、細胞源の探索とその評価は引き続き重要な検討項目である。

本研究では、遺伝子工学的手法により遺伝子導入を行い、高機能発現を達成した肝がん由来細胞株(ヘパトーマ細胞)に着目した。一般的にヘパトーマ細胞は旺盛な増殖能を有しているが肝機能が極めて低いという問題がある。これに対し、本研究において着目した遺伝子導入マウスヘパトーマ(Hepa/8F5)細胞は、マウス由来ヘパトーマ Hepa1-6 細胞に対し、8 種類の肝特異的転写因子遺伝子を導入した細胞である。遺伝子導入に際し、テトラサイクリン遺伝子発現誘導系を利用することにより Doxycycline (Dox)の添加時のみ肝転写因子が活性化され、細胞増殖の停止と肝特異的機能が発現可能な細胞株として樹立された細胞株である。Hepa/8F5 細胞は無限増殖能を有しているため、細胞の大量供給が可能である。さらに、遺伝子発現誘導剤として Dox を培地中に添加することにより、細胞増殖を停止し高い肝機能を発現するという特性を有する。このように Dox の添加により細胞増殖・肝機能発現の On/Off を容易に制御することが可能であるという点から、バイオ人工肝臓の細胞源として期待できるのではないかと考えた。そこで、中空糸を用いた独自の三次元培養法を Hepa/8F5 細胞に適用することにより、Hepa/8F5 細胞の高機能発現に最適な培養法を確立するとともに、その技術を基盤としたバイオ人工肝臓装置の開発に取り組んだ。

第 1 章では、本研究の背景として肝不全患者を取り巻く現状を述べるとともに、本研究の目的及び目的達成のための研究方針を示した。また、本研究のオリジナリティを示し、本研究の位置付けを明確にした。

第 2 章では、急性肝不全とその治療法について概説し、特に本研究のターゲットであるバイオ人工肝臓における既往の研究について述べ、研究上の問題点、解決すべき課題を示した。

第 3 章では、中空糸培養法を Hepa/8F5 細胞に適用することにより、その応答能を評価した。中空糸培養での Hepa/8F5 細胞は Dox の添加に対し、細胞増殖を停止するという応答を示したが、肝特異的機能であるアンモニア除去能は発現されなかった。この結果、さらなる培養条件の最適化の必要性が示された。

第 4 章では、中空糸培養における Hepa/8F5 細胞の高機能発現条件として酸素雰囲気に着目し、酸素分圧が機能発現に与える影響について検討した。この結果、70%酸素分圧下での培養ではHepa/8F5 細胞は、細胞あたりにおいてマウス初代肝細胞と同等以上の肝機能を発現した。また、グルコースと乳酸の代謝状態から細胞内で嫌気的代謝から好気的代謝へと移行していることが示唆された。よって、十分な酸素供給により、Hepa/8F5 細胞は十分な肝機能発現が可能であることが示された。

第 5 章では、前章での検討をもとに、中空糸を用いた Hepa/8F5 細胞培養の至適化に関する検討を行った。まず、酸素要求性を満たすため、中空糸の小口径化(285μm→140μm)を行った。さらに、中空糸内部への播種方法を検討した。この結果、肝特異的機能であるアンモニア除去能、アルブミン分泌能の発現において Hepa/8F5 細胞は初代肝細胞と同等以上の発現が可能であり、通常酸素分圧下での高機能発現が可能な培養条件を見出した。

第 6 章では、これまでの検討を基に、Hepa/8F5 細胞を充填した小動物スケールの中空糸型バイオ人工肝臓装置の開発に取り組んだ。作製した装置を灌流培養下で評価を行った結果、装置単位体積あたりのアンモニア除去能、アルブミン分泌能それぞれの機能発現レベルは同装置に初代肝細胞や胚性幹細胞を充填した場合と同程度を達成した。この結果、Hepa/8F5 細胞を充填したバイオ人工肝臓装置は高レベルでの肝機能発現が可能であることが示された。

第 7 章では、本論文の総括を行うとともに、本研究の今後の課題と展望について述べた。

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