Cellular Engineering of Hepatocyte Cell Lines

描述(申请人提供)：肝细胞移植可以 可能用于治疗肝功能衰竭和基于肝脏的代谢性疾病。 不幸的是，可用于肝细胞分离的人体肝脏数量 移植在整个器官中的使用受到竞争的限制 移植。一种替代原发干细胞移植的潜在方法 肝细胞将使用克隆细胞系，这将提供 可获得性、一致性和无菌优点。这样的细胞可以培养出来 数量不限，且成本远低于隔离的主服务器 肝细胞。我们假设人类肝细胞可以有条件地 永生化和工程化以确保移植治疗的安全性 肝脏疾病。已经产生的大鼠肝细胞系能够 通过定点切除引入的SV40T实现可逆永生化 抗原使用loxP/Cre系统。这种可逆永生化的肝细胞可以 纠正急性和慢性肝功能衰竭动物模型的肝功能 以肝脏为基础的代谢疾病。这项提案将评估可逆的 永生化的人类肝细胞可以成功整合 Rag2-/-小鼠移植后的肝实质 门脉循环。研究还将检查可逆的不朽 人类肝细胞可以起到纠正生理异常的作用 与肝硬变大鼠的慢性肝脏失代偿有关。为了 避免需要病毒基因转移技术，我们还将确定 LoxP/Cre能否产生可逆永生化的人肝细胞 用TAT-Cre融合蛋白诱导重组。最后，这一点 一项提案将评估loxP/Cre产生的人的致瘤潜力 肝细胞。肿瘤抑制机制改变的证据将是 评估和转化分析将被用来直接衡量 永生化人肝细胞切除前后的致瘤潜能。

DESCRIPTION (provided by applicant): Liver cell transplantation could potentially be used to treat liver failure and liver-based metabolic diseases. Unfortunately, the number of human livers available for hepatocyte isolation and transplantation is limited by competition for use in whole organ transplantation. A potential alternative to the transplantation of primary hepatocytes would be to use a clonal cell line, which would provide the advantages of availability, uniformity and sterility. Such cells could be grown in unlimited number and at far less cost compared to isolated primary hepatocytes. We hypothesize that human hepatocytes can be conditionally immortalized and engineered to be safe for transplantation in the treatment of liver diseases. Rat hepatocyte cell lines have been generated that are capable of reversible immortalization by site-specific excision of an introduced SV40 T antigen using the loxP/Cre system. Such reversibly immortalized hepatocytes can correct liver function in animal models of acute and chronic liver failure and liver-based metabolic diseases. This proposal will assess whether reversibly immortalized human hepatocytes can be generated that can successfully integrate into the liver parenchyma of Rag2-/- mice following transplantation through the portal circulation. Studies will also examine whether reversibly immortalized human hepatocytes can function to correct the physiologic abnormalities associated with chronic liver decompensation in cirrhotic rats. In order to avoid the need for viral gene transfer technologies, we will also determine whether loxP/Cre generated reversibly immortalized human hepatocytes can be induced to undergo recombination using a TAT-Cre fusion protein. Finally, this proposal will evaluate the tumorigenic potential of loxP/Cre generated human hepatocytes. Evidence of alterations in tumor suppressor mechanisms will be assessed and transformation assays will be used to directly measure the tumorigenic potential of pre- and post-excision immortalized human hepatocytes.