STUDIES ON THE REPLICATION AND ONCOGENICITY OF HBV

估计有1.76亿慢性B型肝炎病毒携带者 (HBV)世界范围内强有力的流行病学证据将携带者 发展为肝细胞癌。 因此， 研究病毒体外系统的重要性 复制和阐明HBV感染的机制 细胞会发生恶性转化 尽管有可能 通过分离啮齿动物细胞获得病毒功能的部分表达 对于克隆的HBV DNA，这些细胞不支持有效的病毒复制 也不会被恶意转化 然而，克隆的HBV DNA 能够在黑猩猩身上引起肝炎 工作假设 这里提出的实验是，相对弱的启动子 与HBV基因相关，而不是编码表面抗原的基因 病毒的抗原不能克服宿主和/或组织特异性 啮齿动物细胞或非肝来源的细胞施加的限制。 因此，在本发明中， 为了实现我们建立体外研究系统的长期目标， HBV，我们建议1）获得小鼠中病毒基因的表达增加 已经携带整合的HBV基因的细胞系， 已知增强整合的病毒基因组表达的化合物; B） 将他们暴露于肿瘤促进剂和亚有效剂量的物理和 化学致癌物和c）通过“重叠感染”他们与额外的 再环化的HBV基因组或新的杂合质粒，其中病毒 基因组与强启动子或增强序列连接; 2）开发 设计用于在猿细胞中附加体复制或产生 在培养的肝细胞中有效表达病毒功能; 3） 通过引入再环化的 HBV基因组或具有HBV序列的杂合质粒连接到强 启动子导入小鼠胚胎细胞; 4）开发原位 适合与活组织检查材料一起使用杂交 HBV基因表达与慢性乙型肝炎不同临床分期的关系 肝脏疾病 这些研究应该至少阐明一些因素 在转基因小鼠中， 和受感染个体的肝脏中。

There are an estimated 176 million chronic carriers of hepatitis B virus (HBV) world wide and strong epidiomological evidence linking the carrier state with development of hepatocellular carcinoma. Thus, it is of utmost importance to have in vitro systems available for studying viral replication and for elucidating the mechanism(s) by which HBV infected cells become malignantly transformed. Although it has been possible to obtain partial expression of viral functions by transfecting rodent cells with cloned HBV DNA, these cells do not support efficient viral replication nor do they become malignantly transformed. Nevertheless, cloned HBV DNA is capable of causing hepatitis in chimpanzees. The working hypothesis for the experiments proposed here is that the relatively weak promoters associated with HBV genes other than the gene coding for the surface antigen of the virus cannot overcome host and/or tissue specific restrictions imposed by rodent cells or cells of non-hepatic origin. Thus, to meet our long-term goal of establishing in vitro systems for studying HBV, we propose 1) to obtain increased expression of viral genes in mouse lines that already carry integrated HBV genes by a) treating them with compounds known to enhance expression of integrated viral genomes; b) exposing them to tumor promoters and subeffective doses of physical and chemical carcinogens and c) by "superinfecting" them with additional recircularized HBV genomes or with new hybrid plasmids in which the viral genome is linked to strong promoters or enhancing sequences; 2) to develop hybrid plasmids designed to replicate episomally in simian cells or to give efficient expression of viral functions in cultured hepatocytes; 3) to create transgenic mice carrying HBV sequences by introducing recircularized HBV genomes or hybrid plasmids with HBV sequences linked to strong promoters into mouse embryo cells; 4) to develop methods of in situ hybridization suitable for use with biopsy material which will allow association of HBV gene expression with various clinical stages of chronic liver disease. These studies should elucidate at least some of the factors that regulate expression of HBV genes in cultured cells, in transgenic mice and in the liver of infected individuals.