Bioartificial liver development using reversibly immortalized human liver cell lines.

急性肝功能衰竭（ALF）通常危及生命，并显著降低患者的生活质量。原位肝移植已成为治疗ALF的一种成功方法，但该方法成本高，受供体肝稀缺的限制，并且与高发病率和死亡率相关。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（巴尔斯）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的技术已经大大进步，目前正在研究巴尔斯的许多设计，包括（1）生物组件，（2）生物反应器，和（3）全血或血浆灌注系统。不太可能以足以治疗超过一部分需要生物人工肝（BAL）治疗的患者的规模分离人肝细胞。动物细胞的使用导致了与感染性病原体的传播以及供体和人类之间的免疫和生理不相容性相关的问题。人胚胎干细胞和骨髓多能成体祖细胞作为巴尔斯的可能来源受到了极大的关注。使用严格调控的克隆肝细胞系将是有吸引力的。这样的细胞系在组织培养中经济地生长，并提供均匀性、无菌性和无病原体的优点。本研究以Cre/loxP介导的位点特异性重组技术为基础，利用可逆永生化的人肝细胞构建了巴尔斯，并取得了以下成果：1）p21转导增强了肝细胞的分化功能; 2）利用逆转录病毒载体SSR#69构建了人肝内皮细胞系，该载体编码侧接loxP的猿猴病毒40大T抗原cDNA; 3）建立了从手术切除的猪肝段中分离肝细胞的方法。

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.