Study of liver specific gene expression in primary cultured hepatocytes for establishing hybrid Artificial liver

对于混合型人工肝来说，建立一种维持肝脏特异性功能的培养系统是必不可少的。用Northern杂交技术检测原代培养的大鼠肝细胞中肝脏特异性基因(白蛋白和鸟氨酸转氨酶)的表达。在开始培养的24小时内，该基因的表达水平显著下降，并且在不同培养条件下仍保持较低的表达水平。相反，生长相关基因(癌基因和鸟氨酸脱羧酶)的表达在开始培养后48小时内增加。这些结果表明，原代培养中肝脏特异性基因表达的下调是与细胞增殖密切相关的去分化过程。为了进一步研究肝细胞生长刺激的机制，分析了导致c-myc表达的信号转导途径。我们发现，抑制磷脂酰肌醇的转换以剂量依赖的方式显著减弱c-myc的表达，而细胞内钙离子的补充逆转了这一效应。提示在培养的肝细胞中，加速的磷脂酰肌醇转换和Ca~(2+)&Gt~(2+)动员维持c-myc的恒定表达。需要对培养的肝细胞进行持续生长刺激的进一步分析，以建立可能维持肝脏特异性功能的生长停滞细胞。

It is essential for a hybrid artificial liver to establish a culture system which maintains liver-specific function. Expression of liver-specific genes (albumin and ornithine transcarbamylase) in rat hepatocytes in primary culture was examined by Northern blot hybridization. The expression was significantly decreased within 24 hours after starting the culture and the low level of the expression sustained under various culture conditions. In contrast, growth related gene expression (oncogenes and ornithine decarboxylase) increased within 48 hours after starting the culture. These results indicate that the depression of liver specific gene expression in the primary culture is de-differentiation process which is closely linked with cell proliferation. To further examine the mechanism of growth stimulation in the cultured hepatocytes, signal transduction pathways leading to c-myc expression were analyzed. We found that inhibition of phosphatidylinositol turnover significantly attenuated the c-myc expression in a dose-dependent manner and that supplement of intracellular Ca^<2+> reversed this effect. It was thereby suggested that accelerated phosphatidylinositol turnover and Ca^<2+> mobilization maintains constant expression of c-myc in the cultured hepatocytes. Further analysis of persisting growth stimulation in cultured hepatocytes is required to establish growth arrested cells which may maintain the liver specific functions.