Molecular Analysis of Stem Cell in Hepatocarcinogenesis

采用退化引物同源克隆的方法比较了RTK基因在大鼠正常肝脏和二乙基亚硝胺诱导的肝癌组织中的表达谱。与正常肝脏相比，Tie-2、c-Met和Flk-1基因是大鼠肝癌中克隆量最多的RTK基因。原位杂交和免疫组织化学研究显示c-Met和Flk-1在GST-P阳性的癌前病变和肿瘤病变中过表达。Tie-2不仅在内皮细胞中表达，也在所谓的卵圆细胞中表达，卵圆细胞被认为是肝干细胞。在正常肝脏和肝癌细胞/组织中均检测到Tie-2配体血管生成素-1 mRNA。相反，血管生成素-2 mRNA仅在肝癌组织中检测到。我们接下来的目的是研究半胱天冬酶和钙蛋白酶在h2o2诱导的肝细胞凋亡中的作用。在250 ~ 1000μ m范围内，tunel阳性细胞凋亡与H2O2处理下的聚adp -核糖聚合酶（PARP）裂解和原aspase-3蛋白水解呈剂量依赖性平行发生。H2O2浓度为bb0 ~ 250μM时，Bcl-xL和完整Bax表达水平降低。裂解形式的Bax出现在caspase-3激活之前，并以剂量依赖性的方式增加。这些结果表明，Bax切割是肝细胞caspase依赖性凋亡的上游信号。Bax裂解的分子分析可能揭示肝癌细胞发生的机制。最后，我们感谢这项资助的支持以及我们博士后研究员和工作人员的宝贵贡献。

We compared the expression profile of RTK genes in rat normal liver and diethylnitrosamine-induced hepatoma tissues using a homology cloning method with degenerated primers. The Tie-2, c-Met, and Flk-1 genes were the most abundant RTK genes cloned in rat hepatoma compared to normal liver. In situ hybridization and immunohistochemical studies showed overexpression of c-Met and Flk-1 in GST-P positive preneoplastic lesions as well as neoplastic lesions. Tie-2 was expressed not only in endothelial cells but also in so-called oval cells, which are thought to be liver stem cells. Tie-2 ligand, angiopointin-1, mRNA was detected in both normal livers and hepatoma cells/tissues. In contrast, angiopoietin-2 mRNA was detected only in hepatoma tissues.We next aimed to examine the involvement of caspases and calpains in H2O2-induced hepatic cell apoptosis. TUNEL-positive apoptotic cells appeared in parallel with poly(ADP-ribose) polymerase (PARP) cleavage and procaspase-3 proteolysis by H2O2 treatment in a dose-dependent manner (250-1000μM). Bcl-xL and intact Bax expression levels decreased when H2O2 was >250μM. The cleaved form of Bax appeared prior to caspase-3 activation, increasing in a dose-dependent manner. These results indicate that Bax cleavage is upstream signal of caspase-dependent apoptosis in hepatocytes. Molecular analysis of Bax cleavage may allow the mechanism of development of hepatoma cell.Finally, we acknowledge a support from this grant and a valuable contribution of our post doc fellows and staffs.