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Molecular mechanism of hepatic carcinogenesis and hepatocyte apoptosis in the Rb transgenic mice

Rb转基因小鼠肝癌发生及肝细胞凋亡的分子机制

基本信息

批准号：
15390393
负责人：
MIURA Naoyuki
金额：
$ 9.22万
依托单位：
Hamamatsu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

We created the Rb transgenic mice in which the human Rb cDNA was controlled under the HNF-1 promoter/enhancer and got two lines. We determined that the A mouse had 11 copies of the transgene per haploid and the B mouse had 4 copies per haploid.We injected diethylnitrosamine into the abdominal cavity of the A mice, B mice and wild-type mice and then feed them with phenobarbital-containing water for 35 weeks. After sacrifice, the livers were examined. In the wild-type mice, several hepatocellular carcinomas were detected while no carcinomas were detected in the Rb transgenic mice, A and B mice. We extracted DNAs from the hepatocellular carcinoma in the wild-type mice and investigated the mutations of Rb,p53, Ras and Myc genes, but no mutations were detected. The number of nodules in the liver was largest in the wild-type mice, secondarily larger in the B mice and lesser in the A mice. This result indicates that Rb protein inhibits nodule formation in a dose-dependent manner.To find the molecules involved in resistance to fulminant hepatitis, firstly the cellular extracts from the livers were prepared and subjected to Western blotting. The results showed that the amounts of caspase 1, caspase 3,p53,E2F1-E2F5,Bcl-2,Bcl-XL,Bcl-XS, Bad and Bid proteins showed no differences between the wild-type and A mice. However, the Bax protein was decreased in the A mice and B mice compared to that in the wild-type mice. Second, we did 2-dimensional electrophoresis using the protein extracts from the livers in the A mice and wild-type mice. We found that 5 proteins appeared in the A mice, but not in the wild-type mice and 7 proteins disappeared in the A mice, but not in the wild-type mice.

我们建立了人Rb基因在HNF-1启动子/增强子调控下的转基因小鼠，并获得了两个品系。我们确定A小鼠每个单倍体有11个转基因拷贝，B小鼠每个单倍体有4个拷贝，我们将二乙基亚硝胺注射到A小鼠、B小鼠和野生型小鼠的腹腔中，然后用含苯巴比妥的水喂养它们35周。处死后，检查肝脏。在野生型小鼠中，检测到几种肝细胞癌，而在Rb转基因小鼠、A和B小鼠中未检测到癌。我们从野生型小鼠肝癌组织中提取DNA，检测Rb、p53、Ras和Myc基因的突变，但未检测到突变。野生型小鼠的肝脏结节数量最多，其次是B小鼠，A小鼠较少。这一结果表明Rb蛋白以剂量依赖的方式抑制结节的形成。为了寻找参与抵抗暴发性肝炎的分子，首先制备了来自肝脏的细胞提取物并进行了Western印迹。结果表明，caspase 1、caspase 3、p53、E2 F1-E2 F5、Bcl-2、Bcl-XL、Bcl-XS、Bad和Bid蛋白的表达量在野生型小鼠和A小鼠之间没有差异。然而，与野生型小鼠相比，Bax蛋白在A小鼠和B小鼠中减少。第二，我们使用A小鼠和野生型小鼠肝脏的蛋白质提取物进行二维电泳。我们发现有5种蛋白质在A小鼠中出现，但在野生型小鼠中没有，7种蛋白质在A小鼠中消失，但在野生型小鼠中没有。