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

通过转基因技术建立丙型肝炎和肝细胞癌动物模型

基本信息

批准号：
08457077
负责人：
ESUMI Mariko
金额：
$ 4.35万
依托单位：
Nihon University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

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.

1.转基因小鼠的制备为了建立丙型肝炎和肝细胞癌的动物模型，我们制备了31只携带丙型肝炎病毒（HCV）基因组的转基因小鼠：携带部分基因组（包括白蛋白启动子/增强子控制下的结构蛋白区域）的3系转基因小鼠8）、19个具有HCV全基因组的株系（Alb-HN 2）和9个具有SR α启动子控制下的全基因组的株系（ME-HN 2）。HCV特异性RNA在出生后8周内几乎不表达.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。甲基化敏感的限制性内切酶片段分析和甲基化胞嘧啶的亚硫酸氢盐基因组测序的映射表明，转基因广泛甲基化。去甲基化剂，5-氮杂胞苷，诱导HCV基因表达的6行检查中的4。这些结果提示HCV基因甲基化是其在转基因小鼠中表达受抑制的原因之一.转基因小鼠肝脏的组织学变化我们观察了转基因小鼠肝脏的长期病理变化。在7至22月龄的小鼠中观察到肝炎，如暴发性肝炎的半急性期和伴有淋巴细胞浸润和腹水积聚的肝萎缩：Alb-HN 3.8- 1系1只小鼠、Alb-HN 2 -48系4只小鼠中的1只和ME-HN 2 -5系6只小鼠中的1只。肝组织中检测到HCVRNA的表达。对于品系Alb-HN 2 -44，在17个转基因肝脏中的2个中观察到发育异常变化。其中1例发生腺瘤。这些结果表明，只有转基因的表达才能引起肝脏的病理变化。然而，有必要进一步检查观察到的这些变化是否与转基因相关。