Bioartificial liver development using reversibly immortalized human liver cell lines.

使用可逆永生化人类肝细胞系开发生物人工肝。

• 批准号: 13470236
• 负责人: TANAKA Noriaki
• 金额: $ 10.56万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470236/
• 关键词: reversible immortalization; human hepatocyte cell line; human liver endothelial cell line; bioartificial liver; porcine hepatocyte isolation; p21; 不死化肝細胞; テロメレース; ブタ肝不全

Acute liver failure (ALF) is often life-threatening and dramatically diminishes the quality of life of patients. Orthotopic liver transplantation has become a successful therapy of ALF, but this procedure is highly costly, limited by the scarcity of donor livers and associated with high morbidity and mortality. There is a compelling need for developing effective alternatives for patients with ALF. Considering the potential of the liver to regenerate, temporary support with bioartificial livers (BALs) is an attractive approach. Since technologies of tissue cell culture and biomaterials have been greatly advanced, many designs of BALs, including (1) a biological component, (2) a bioreactor, and (3) a whole blood or plasm a perfusion system are currently under investigating. It is unlikely that human hepatocytes can be isolated on a scale sufficient to treat more than a fraction of the patients who need bioartificial liver (BAL) treatment. The use of animal cells results in the concerns related to the transmission of infectious pathogens and immunologic and physiologic incompatibilities between the donor and humans. Human embryonic stem cells and bone marrow multipotent adult progenitor cells have receive great attention as a possible source for BALs. The use of tightly regulated clonal hepatocyte cell lines would be attractive. Such cell lines grow economically in tissue culture and provide the advantage of uniformity, sterility, and freedom of pathogens. In this project, we have made great efforts to develop BALs using reversibly immortalized human liver cells based on Cre/loxP-mediated site-specific recombination and achieved the following results :1) Enhancement of hepatic differentiated functions with p21 transduction,2) Establishment of a human liver endothelial cell line using a retroviral vector SSR#69 encoding a loxP-flanked simian virus 40 large T antigen cDNA,3) Establishment of hepatocyte isolation method from a surgically resected porcine hepatic segment.

急性肝衰竭（ALF）往往危及生命，并显著降低患者的生活质量。原位肝移植已成为一种成功的治疗ALF的方法，但由于供体肝脏的稀缺以及高发病率和死亡率，该手术成本高昂。迫切需要为ALF患者开发有效的替代方案。考虑到肝脏再生的潜力，生物人工肝脏（BALs）的临时支持是一种有吸引力的方法。由于组织细胞培养和生物材料技术的进步，目前正在研究许多生物燃料的设计，包括(1)生物成分，(2)生物反应器，(3)全血或全浆灌注系统。人类肝细胞的分离规模不太可能足以治疗一小部分需要生物人工肝（BAL）治疗的患者。动物细胞的使用引起了与传染性病原体的传播以及供体与人之间的免疫和生理不相容有关的问题。人胚胎干细胞和骨髓多能成体祖细胞作为BALs的可能来源而受到广泛关注。使用严格调控的克隆肝细胞细胞系将是有吸引力的。这种细胞系在组织培养中经济地生长，并具有均匀性、无菌性和不受病原体影响的优点。在这个项目中，基于Cre/ loxp介导的位点特异性重组，我们利用可逆永生化的人肝细胞开发了BALs，并取得了以下成果：1)通过p21转导增强肝脏分化功能；2)利用编码loxp侧翼猿猴病毒40大T抗原cDNA的逆转录病毒载体SSR#69建立了人肝内皮细胞系；3)建立了从手术切除的猪肝脏中分离肝细胞的方法段。

项目成果

Kunieda T. et al.: "Transduction of immortalized human hepatocytes with p21 to enhance differentiated phenotypes"Cell Transplant. 11. 421-428 (2002)

Kunieda T. 等人：“用 p21 转导永生化人肝细胞以增强分化表型”细胞移植。

T. Watanabe, N. Shibata, K. A. Westerman, T. Okitsu, J. E. Allain, M .Sakaguchi, T. Totsugawa, M. Maruyama, T. Matsumura, H. Noguchi, S. Yamamoto, M. Hikida, A. Ohmori, M. Reth, A. Weber, N. Tanaka, P. Leboulch, N. Tanaka, N. Kobayashi.: "Establishment of

T. Watanabe、N. Shibata、K. A. Westerman、T. Okitsu、J. E. Allin、M.Sakaguchi、T. Totsukawa、M. Maruyama、T. Matsumura、H. Noguchi、S. Yamamoto、M. Hikida、A. Ohmori、

Watanabe T, et al.: "Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers"Transplantation. (in press). (2003)

Watanabe T 等人：“建立永生化人肝星状清道夫细胞以开发生物人工肝”移植。

H. Noguchi, N. Kobayashi, K. A. Westerman, M. Sakaguchi, T. Okitsu, T. Totsugawa, T. Watanabe, T. Matsumura, T. Fujiwara, T. Ueda, N. Tanaka, P. Leboulch.: "Controlled Expansion of Human Endothelial Cell Populations by Cre-loxP-Based Reversible Immortaliz

H. Noguchi、N. Kobayashi、K. A. Westerman、M. Sakaguchi、T. Okitsu、T. Totsukawa、T. Watanabe、T. Matsumura、T. Fujiwara、T. Ueda、N. Tanaka、P. Leboulch。：“受控

N. Kobayashi, K. A. Westerman, N. Tanaka, I. J. Fox, P. Leboulch.: "A reversibly immortalized human hepatocyte cell line as a source of hepatocyte-based biological support. (Review)"Addiction Biology. 6. 293-300 (2001)

N. Kobayashi、K. A. Westerman、N. Tanaka、I. J. Fox、P. Leboulch.：“作为基于肝细胞的生物支持来源的可逆永生化人类肝细胞系。（评论）”成瘾生物学。