TRANSGENIC MOUSE MODEL FOR STUDY OF HEPATITIS C

尽管丙型肝炎病毒（HCV）是世界范围内发病率和死亡率的主要原因，但病毒基因表达对感染细胞的体内影响尚不清楚。在此之前，我们报道了表达HCV结构蛋白（core， E1和E2）的转基因小鼠的构建，并表明HCV结构蛋白的表达在该动物模型中不直接引起细胞病变。利用DNA免疫，我们能够在这些转基因动物中诱导针对HCV核心的抗体、T细胞增殖和细胞毒性T细胞反应，但仅针对包膜蛋白的抗体。相反，细胞免疫反应似乎主要针对野生型小鼠的包膜蛋白。这些结果表明，野生型小鼠对HCV结构蛋白（包膜>核心）的CTL反应是有层次的，而转基因小鼠对核心的免疫无知，但在T细胞水平上对包膜蛋白耐受。本实验室也培育了表达HCV全长多蛋白的转基因小鼠，并正在开发HCV转基因诱导表达系统。在四环素调控系统的基础上，我们建立了SV40 T抗原（TAg）和LacZ两种转基因基因在肝脏中的条件表达可通过四环素严格调控的二元转基因模型。利用小鼠白蛋白或小鼠尿蛋白（MUP）启动子在一组转基因小鼠中实现了四环素应答转录激活子（tTA）的肝脏特异性表达。这些小鼠与携带TAg或LacZ的转基因小鼠在ta调控启动子的控制下杂交。对tTA和TAg（或LacZ）转基因小鼠的分析表明，转基因基因的肝脏特异性表达可以被抑制到不可检测的水平，并在四环素给药和停药的可逆方式下受到调节。tTA和TAg转基因小鼠发生肝细胞腺瘤和肝细胞增生，连续给药四环素可预防。该实验证明了这种二元转基因模型在肝脏特异性研究任何潜在基因的生理功能方面的价值。我们目前正在使用该模型系统评估HCV蛋白在肝脏中的调控表达。这些动物将提供一个有用的动物模型，不仅可以解决HCV基因产物的免疫发病机制和细胞病变潜力问题，还可以研究HCV在体内的复制。

Although hepatitis C virus (HCV) is a leading cause of morbidity and mortality worldwide, the effects of viral gene expression on infected cells remain unclear in vivo. Previously, we reported the construction of transgenic mice expressing HCV structural proteins (core, E1 and E2) and showed that expression of HCV structural proteins is not directly cytopathic in this animal model. Using DNA immunization, we were able to induce antibodies, T cell proliferative and cytotoxic T cell responses against the HCV core but only antibodies against the envelope protein in these transgenic animals. In contrast, the cellular immune responses appeared to target predominantly against the envelope proteins in wild-type mice. These results suggest a hierarchy of CTL response against the HCV structural proteins (envelope>core) in the wild-type mice, and an immunological ignorance toward the core but tolerance toward the envelope proteins at the T cell level in the transgenic mice. Our laboratory has also generated transgenic mice expressing HCV full-length polyprotein and is developing a system for the inducible expression of HCV transgene Based on the tetracycline regulatory system, we established a binary transgenic model in which the conditional expression of two transgenes, SV40 T antigen (TAg) and LacZ, can be tightly regulated in the liver by administration of tetracycline. Mouse albumin or mouse urinary protein (MUP) promoter was used to achieve liver-specific expression of the tetracycline-responsive transcriptional activator (tTA) in one set of transgenic mice. These mice were crossed with transgenic mice carrying either TAg or LacZ under the control of tTA-regulated promoter. Analyses of mice transgenic for both tTA and TAg (or LacZ) revealed that the liver-specific expression of the transgenes could be suppressed to undetectable level and regulated in a reversible fashion by tetracycline administration and withdrawal. Mice with tTA and TAg transgenes developed hepatocellular adenomas and hyperplasia that could be prevented by continuous tetracycline administration. This experiment demonstrates the value of this binary transgenic model in studying the physiological functions of any potential genes of interest in a liver-specific manner. We are currently evaluating the regulated expression of HCV proteins in the liver using this model system. These animals will provide a useful animal model not only to address issues of immunopathogenesis and cytopathic potential of HCV gene products but also to study HCV replication in vivo.