黄曲霉毒素B1转化体外原代培养的大鼠肝上皮细胞

利用基因转染技术，研究黄曲霉毒素B1在人c-myc基因和p450IA2基因转染的基础上，转化体外原代大鼠脉上皮细胞的可能性及其部分分子机理。首先构建Xm-6／c-myc真核表达载体，并将其转染Alexander细胞，免疫组化实验证明重组的c-mvc基因可在肝细胞中表达。随后分别将c-myc和人p450IA2表达载体转染无血清原代培养的新生Wistar大鼠肝细胞，结果发现p450IA2基因可代谢活化5ng／ml的AFB1，并且在外源c-myc基因辅助下，诱导大鼠肝上皮细胞获得生长优势。其中随机挑选的1例在渡过长达4个月的“危机”期后，获得永生化生长能力。从CK-18和大鼠白蛋白的表达情况和细胞超微结构特征，明确了转化细胞来源于肝上皮细胞。核型分析、免疫组化和Northern杂交分析提示，大鼠肝上皮细胞转化与其基因组稳定性改变和TGFα高表达相关。

Transformation of Rat Hepatocytes in an in vitro Primary Culture by Aflatoxin B1

Aflatoxin B1 (AFB1) is one of the major causative factors of hepatocellular carcinoma.In this study, the combined effects of AFB1 activated by human cytochrome p45O IA2 and cmyc in transformation of rat hepatocytes were investigated in an in vitro primary culture system. The expression vectors, Xm-6/c-myc was first constructed and their expression possibilities were examined in Alexander cells by immunocytochemistry. Then both c-myc and human cytochrome p450 IA2 expression vectors were sequentially trans fected into newborn rat liver cells in serum-free primary culture. Results showed that p45O IA2 could activate AFB1 at concentrations as low as 5 ng/ml, and the activated AFB1 coupled with exogenous c-myc could induce rat hepatocytes to survive and grow beyond two-month limit in primary culture.During long-term in vitro culturing including four-month in crisis, one of the randomly selected transformed hepatocytes with the growth advantage became immortalized. Immunocytochemical assays for CK-l8 and rat albumin plus observed electron microscopic features clearly confirmed these cells derived from epithelial hepatocytes. Further characterization showed that the pro ss of immortalization was associated with chromosomal abnormalities and elevated expression of TGFa.