Molecular investigation on the fulminant hepatitis-resistant model animals

在大鼠肝细胞核因子-1(HNF-1)启动子/增强子启动子/增强子的KBP控制下，获得了两株人Rb基因的转基因小鼠。转基因小鼠A系(TGA)每单倍体约有11个拷贝，B系(TGB)约有4个拷贝。经Western印迹分析，TGA小鼠肝脏中有大量Rb蛋白表达，TGB小鼠肝脏中有少量Rb蛋白表达。对照组小鼠注射抗Fas抗体和肿瘤坏死因子α后，血清谷草转氨酶和谷丙转氨酶升高，肝组织出现出血和肝细胞凋亡。而TGA小鼠GOT、GPT轻度升高，未见出血和细胞凋亡，GOT、GPT中度升高，大量肝细胞发生凋亡，未见出血。为了探讨抗细胞凋亡的分子机制，我们对…进行了蛋白质印迹分析。更多与细胞凋亡相关的蛋白质。Fas、Bcl2、BclX、Bid、Bad、ICE、CPP32、E2F1、E2F2、E2F3、E2F4、E2F5和P53蛋白在对照组和Rb转基因小鼠中无差异。然而，与对照小鼠相比，Rb转基因小鼠的Bax蛋白表达减少。这表明Bax蛋白是抗凋亡条件下的贡献蛋白之一。接下来，我们研究了Rb转基因小鼠是否对化学致癌具有抵抗力。我们在6周龄时将二乙基亚硝胺注射到腹膜腔内，并在饮用水中用苯巴比妥治疗35周。实验结束后，处死小鼠，制作组织切片，计数肝细胞癌和肝结节数目。对照组小鼠出现了大量的结节和几个肝细胞癌。相比之下，Rb转基因小鼠的结节数量大大减少，未检测到肝细胞癌。这些结果表明，Rb蛋白在体内具有抗肝细胞凋亡剂和抗肿瘤作用。较少

Two lines of the transgenic mice in which the human Rb cDNA was controlled under the) kbp of rat hepatocyte nuclear factor-1 (HNF-1) promoter/enhancer were generated. Transgenic mice, line A (TGA) had about 11 copies of the transgenes per haploid and line B (TGB) had about 4 copies of the transgenes. By Western blot analysis, we observed that a large amount of Rb protein was expressed in the liver of TGA mice and a small amount of Rb protein was expressed in that of TGB mice. In control mice, injection of anti-Fas antibody and TNFα induced increase of GOT and GPT in serum, hemorrhages and hepatocyte apoptosis in the histology. However, in TGA mice, the increase in GOT and GPT was marginal and no hemorrhages and apoptosis were detected In TGB mice, the increase in GOT and GPT was moderate and apoptosis of the substantial number of hepatocytes was observed, but no hemorrhages. In order to investigate the molecular mechanism of anti-apoptotic condition, we did the western blot analysis of … More apoptosis-related proteins. Fas, Bcl-2, Bcl-X, Bid, Bad, ICE, CPP32, E2F1, E2F2, E2F3, E2F4, E2F5 and p53 proteins had no differences between control mice and Rb transgenic mice. However, the Bax protein was decreased in Rb transgenic mice compared to control mice. This suggests that the Bax protein is on e of the contributing proteins for the anti-apoptotic condition.Next we investigated whether the Rb transgenic mice showed resistance to chemical carcinogenesis. We inject diethylnitrosamine into the peritoneal cavity at the age of 6 weeks and treated phenobarbital in drinking water for 35 weeks. After the experiment, the mice were sacrificed to make histological sections for counting the numbers of hepatocellular carcinoma and hepatic nodule. In control mice, a large number of nodules and several hepatocellular carcinoma were developed. In contrast, the number of nodules was greatly reduced and no hepatocellular carcinomas were detected in Rb transgenic mice. These results indicate that the Rb protein act as an anti apoptotic agent and an anti-tumorigenic agent in the liver in vivo. Less