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.

我们创建了RB转基因小鼠，其中人Rb cDNA在HNF-1启动子/增强子下受到控制，并获得了两条线。我们确定A小鼠的每单倍体中有11份副本，而B小鼠的每倍倍倍4个副本。我们在A小鼠，B小鼠和野生型小鼠的腹腔中注射二乙基硝基胺含有水35周。牺牲后，检查了肝脏。在野生型小鼠中，在RB转基因小鼠A和B小鼠中检测到几只肝细胞癌。我们从野生型小鼠的肝细胞癌中提取了DNA，并研究了RB，p53，Ras和Myc基因的突变，但未检测到突变。在野生型小鼠中，肝脏中的结节数量最大，其次在B小鼠中较大，而在A小鼠中则较小。该结果表明，RB蛋白以剂量依赖性抑制结节形成。要找到与暴发性肝炎抗性有关的分子，首先制备了来自肝脏的细胞提取物并进行蛋白质印迹。结果表明，caspase 1，caspase 3，p53，e2f1-e2f5，bcl-2，bcl-xl，bcl-xl，bcl-xs，坏蛋白的量在野生型和小鼠之间没有差异。然而，与野生型小鼠相比，A小鼠和B小鼠的Bax蛋白减少了。其次，我们使用来自A小鼠和野生型小鼠的肝脏的蛋白质提取物进行了二维电泳。我们发现在A小鼠中出现了5种蛋白质，但在野生型小鼠中没有出现，而在A小鼠中则消失了7种蛋白质，而在野生型小鼠中则不消失。