Development of animal models with hepatitis C and hepatocellular carcinoma by transgenic techniques

1. 为了建立丙型肝炎和肝细胞癌的动物模型，我们培育了31只携带丙型肝炎病毒（HCV）基因组的转基因小鼠：3系携带白蛋白启动子/增强子控制的包括结构蛋白区域在内的部分基因组（Alb-HN3.8）， 19系携带HCV全基因组（Alb-HN2）， 9系携带SRalpha启动子控制的全基因组（ME-HN2）。出生后8周内hcv特异性RNA表达很少。为了分析转基因失活的机制，我们检测了转基因的甲基化失活。甲基化敏感限制性内切酶片段分析和亚硫酸氢盐基因组测序的甲基化胞嘧啶图谱显示，该转基因基因被广泛甲基化。一种去甲基化剂，5-氮杂胞苷，在检测的6个品系中有4个品系诱导HCV基因表达。这些结果表明HCV cDNA甲基化是其在转基因小鼠中抑制表达的原因之一。我们研究了转基因小鼠肝脏的长期病理变化。在7 ~ 22月龄小鼠中观察到暴发性肝炎半急性期、肝萎缩伴淋巴细胞浸润和腹水积存等肝炎：alb - hn3.8 - 1系1只、Alb-HN2-48系4只、ME-HN2-5系6只中各有1只。在这些肝组织中检测到HCV RNA的表达。至于Alb-HN2-44系，17个转基因肝脏中有2个观察到发育异常。其中一人得了腺瘤。这些结果表明，只有转基因基因的表达才会引起肝脏的病理改变。然而，这些变化是否与转基因有关还有待进一步研究。

1. Production of transgenic miceTo establish an animal model of hepatitis C and hepatocellular carcinoma, we generated 31 transgenic mice carrying hepatitis C virus (HCV) genome : 3 lines of transgenic mice carrying a partial genome including the structural protein region under the control of the albumin promoter/enhancer (Alb-HN3.8), 19 lines with the whole genome of HCV (Alb-HN2) and 9 lines with the whole genome under the control of the SRalpha promoter (ME-HN2). HCV-specific RNA was little expressed within 8 weeks after birth.2. Induction of transgene expressionTo analyze the mechanism of transgene inactivation, we examined methylation inactivation of the transgene. Methylation-sensitive restriction enzyme fragment analysis and mapping of methylated cytosine by bisulfite-genome sequencing showed that the transgene was extensively methylated. A demethylating agent, 5-azacytidine, induced HCV gene expression in 4 of 6 lines examined. These results suggest that methylation of HCV cDNA is a cause of its suppressive expression in transgenic mice.3. Histopathological changes of transgenic liverWe investigated long-term pathological changes in liver of transgenic mice. Hepatitis such as semi-acute phase of fulminant hepatitis and liver atrophy with lymphocyte infiltration and ascitic accumulation was observed in mice with 7 to 22 months of age : I of 1 mouse in line Alb-HN3.8-l, 1 of 4 mice in line Alb-HN2-48, and 1 of 6 mice in line ME-HN2-5. The expression of HCV RNA was detected in these liver tissues. As for line Alb-HN2-44, dysplastic changes were observed in 2 of 17 transgenic livers. One of them developed adenoma. These results suggest that only the expression of transgene induces pathological change of liver. However, it is further necessarily examined whether these changes observed are transgene-associated.