CELLULAR ENGINEERING OF HEPATOCYTE CELL LINES

描述：(改编自申请者的摘要)尽管 与全肝移植相关的成功，手术风险 和并发症大大增加了患者的发病率和 死亡率。肝细胞移植可用于治疗急性肝病 衰竭和基于肝脏的代谢性疾病，并将避免手术 干预及其相关风险。一个潜在的替代方案 原代肝细胞的移植将使用克隆细胞 排队。肝细胞系将提供可用性的优势， 均一、无菌，可无限量、远距离种植 与分离的原代肝细胞相比，成本更低。这项提议将 检查肝细胞是否可以有条件地永生和工程化 在移植给接受者进行治疗时不会产生肿瘤 以肝脏为基础的代谢疾病和肝功能衰竭。为了测试这一点 假说真核表达载体将被构建来控制 SV40大T抗原在转录水平的表达 还有翻译。载体将包含可诱导启动子和 操纵子/抑制子序列。原代肝细胞就会永生 通过转染这些构建物，细胞将在 用于确定基因表达的许可和非许可条件 他们的分化功能水平。基因转录调控 以及各种条件下的移位将通过以下方式进行评估 检测SV40T抗原mRNA和蛋白的产生及细胞生长情况 在体外和免疫缺陷小鼠移植后进行评估。在……里面 为了提供另一种方法来保护可能的异常细胞 在受体中生长，有条件的永生化细胞将被转导到 表达一种自杀基因。介绍单纯疱疹病毒- 胸苷激酶基因将为消除移植的 细胞，如果需要，用更昔洛韦处理。有条件永垂不朽 然后将肝细胞移植到NAGASE蛋白血症大鼠和大鼠体内 用醋酸铵诱导的肝昏迷来评估这些 在体内纠正肝功能缺陷的细胞。总而言之，这些 研究将确定有条件永生化的肝细胞 可以将这些线路设计为不会致癌，并且在 治疗代谢和全身性肝功能不全。

DESCRIPTION: (Adapted from the applicant's abstract) Despite growing successes associated with whole organ liver transplantation, surgical risks and complications contribute significantly to patient morbidity and mortality. Hepatocyte transplantation could be used to treat acute liver failure and liver-based metabolic diseases and would avoid surgical intervention and its associated risks. A potential alternative to the transplantation of primary hepatocytes would be the use of a clonal cell line. An hepatocyte cell line would provide the advantages of availability, uniformity and sterility and could be grown in unlimited quantity and at far less cost compared to isolated primary hepatocytes. This proposal will examine whether hepatocytes can be conditionally immortalized and engineered to be non-tumorigenic when transplanted in to recipients for the treatment of liver-based metabolic diseases and liver failure. To test this hypothesis eukaryotic expression vectors will be constructed which control expression of the SV40 large T antigen at the level of both transcription and translation. Vectors will contain inducible promoters and operator/repressor sequences. Primary hepatocytes will then be immortalized by transfection with these constructs and cells will be characterized at the permissive and non-permissive conditions for gene expression to determine their level of differentiated function. Regulation of gene transcription and translocation under the various conditions will be evaluated by measuring SV40 T antigen mRNA and protein production and cell growth will be assessed in vitro and following transplantation in immunodeficient mice. In order to provide one more way to protect against possible abnormal cell growth in recipients, conditionally immortalized cells will be transduced to express a suicide gene. Introduction of the Herpes Simplex Virus - thymidine kinase gene will provide a way of eliminating the transplanted cells if desired by treatment with gancyclovir. Conditionally immortalized hepatocytes will then be transplanted into Nagase aalbuminemic rats and rats with ammonium acetate inducible hepatic coma to assess the ability of these cells to correct deficiencies in liver function in vivo. In summary, these studies will determine whether conditionally immortalized hepatocyte cell lines can be engineered to be non-tumorigenic and safe for use in the treatment of metabolic and global liver deficiencies.