Takehara T, Hayashi N, Miyamoto Y, Yamamoto M, Mita E, Fusamoto H, Kamada T
First Department of Medicine, Osaka University Medical School, Japan.
Hepatology. 1995 Mar;21(3):746-51.
The lack of a small animal model of hepatitis C virus (HCV) infection has impeded elucidation of the pathogenesis of HCV. The aim of this study was to develop an HCV-expressing animal model by means of cationic liposome-mediated in vivo gene transfer. To examine the feasibility of this strategy, pActLacZ, an expression vector composed of the LacZ gene driven by the beta-actin promoter, complexed with lipofectin, was injected retrogradely into the common bile ducts of adult rats. X-Gal histochemical staining clearly showed that the LacZ gene was expressed in hepatocytes, but not in biliary epithelial cells. Maximal expression was observed at a DNA to lipofectin ratio of 1:4. Based on this observation, pAGS3M091, an expression vector containing the full length of HCV complementary DNA (cDNA) preceded by the beta-actin promoter, was evaluated. Two days after in vivo intrabiliary administration of pAGS3M091 complexed with lipofectin, polymerase chain reaction (PCR) amplification of reverse-transcribed liver RNA demonstrated the 5' and 3' portions of HCV transcripts derived from pAGS3M091. Immunohistochemical analysis showed the HCV core protein in a small number of hepatocytes scattered in the hepatic lobules. We conclude that the full-length HCV genome was successfully expressed in adult rat liver by means of cationic liposome-mediated in vivo gene transfer. This model will be useful for determining the immunopathological role of HCV in vivo.
丙型肝炎病毒（HCV）感染的小动物模型的缺乏阻碍了对HCV发病机制的阐明。本研究的目的是通过阳离子脂质体介导的体内基因转移来建立一种表达HCV的动物模型。为了检验该策略的可行性，将由β-肌动蛋白启动子驱动的LacZ基因组成的表达载体pActLacZ与脂质体转染试剂复合后，逆行注射到成年大鼠的胆总管中。X-Gal组织化学染色清楚地表明，LacZ基因在肝细胞中表达，但不在胆管上皮细胞中表达。在DNA与脂质体转染试剂的比例为1:4时观察到最大表达。基于这一观察结果，对含有β-肌动蛋白启动子的HCV互补DNA（cDNA）全长的表达载体pAGS3M091进行了评估。在体内将pAGS3M091与脂质体转染试剂进行胆管内给药两天后，对逆转录的肝脏RNA进行聚合酶链反应（PCR）扩增，证实了源自pAGS3M091的HCV转录本的5'和3'部分。免疫组织化学分析显示在肝小叶中散在的少数肝细胞中有HCV核心蛋白。我们得出结论，通过阳离子脂质体介导的体内基因转移，全长HCV基因组在成年大鼠肝脏中成功表达。该模型将有助于确定HCV在体内的免疫病理作用。
