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

我们建立了在HNF-1启动子/增强子下控制人Rb cDNA的Rb转基因小鼠，得到了两条系。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。分别在A小鼠、B小鼠和野生型小鼠腹腔内注射二乙基亚硝胺，并用含苯巴比妥水喂养35周。献祭后，检查肝脏。在野生型小鼠中检测到几种肝细胞癌，而在Rb转基因小鼠和A、B小鼠中未检测到肝癌。我们从野生型小鼠肝细胞癌中提取dna，研究Rb、p53、Ras和Myc基因的突变，但未检测到突变。野生型小鼠肝脏结节数最多，B型小鼠次之，A型小鼠较少。这一结果表明Rb蛋白以剂量依赖的方式抑制结节的形成。首先制备肝细胞提取物并进行免疫印迹（Western blotting）检测，以寻找抗暴发性肝炎的相关分子。结果表明，caspase 1、caspase 3、p53、E2F1-E2F5、Bcl-2、Bcl-XL、Bcl-XS、Bad和Bid蛋白的表达量在野生型小鼠和A型小鼠中均无差异。然而，与野生型小鼠相比，A型小鼠和B型小鼠的Bax蛋白含量降低。其次，我们利用A型小鼠和野生型小鼠肝脏的蛋白质提取物进行了二维电泳。我们发现有5种蛋白在A型小鼠中出现，而在野生型小鼠中没有出现；有7种蛋白在A型小鼠中消失，而在野生型小鼠中没有出现。

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.