人细胞色素p450 IA2基因的构建及其功能检测──该基因在啮齿类细胞中代谢

本文首先构建了含人类细胞色素ｐ４５０ＩＡ２ｃＤＮＡ的ｐＺｉｐ－ｐ４５０ＩＡ２的真核表达载体，并分别将其转染到Ｖ７９细胞和原代大鼠肝上皮细胞中．Ｎｏｒｔｈｃｒｎ杂交和免疫组化实验显示，Ｖ７９转染细胞中有人ｐ４５０ＩＡ２的ＲＮＡ和蛋白水平的表达。细胞毒实验和ＨＧＰＲＴ位点突变率检测结果证明，外源性ｐ４５０ＩＡ２基因可在Ｖ７９细胞中代谢活化黄曲霉毒素Ｂ１．转染有ｐＺｉｐ－ｐ４５０ＩＡ２质粒的原代大鼠肝细胞可代谢活化５ｎｇ／ｍｌ的黄曲霉毒素Ｂ１．本研究说明了人ｐ４５０ＩＡ２ｃＤＮＡ在ＭＭＬＶ的５′ＬＴＲ系统驱动下，可在体外啮齿类细胞中代谢活化黄曲霉毒素Ｂ１．它为ＡＦＢ１致癌机制的研究提供了一个新的、更为有效的途径。

STABLE EXPRESSION OF HUMAN CYTOCHROME p450 IA2 cDNA AND ITS METABOLIC ACTIVATION OF AFLATOXIN B_1 IN RODENT CELLS IN VITRO

A pZin-p450 IA2 plasmid,an eukaryotic expression vector containing human cytochrome p450 IA2 cDNA,was constructed and then transfected into V79 cell and rat hepatocytes in primary culture. Stable expression of human p450 IA2 cDNA in V79 cells was established by Northern blot analysis and immunocytochemistry. The subsequent assays for cytotoxicity of aflatoxin B1(AFB1)and for its mutational rate in HGPRT locus in V79 cells transfected with pZip-p450 IA2 demonstrated that exogenous p450 IA2 could activate AFB1 in host cell.It was also shown that AFB1 at concentrations as low as 5 ng/ml could be metabolically activated in primary culture of rat hepatocytes in the same manner. It was suggested that this study provided a new effective model to stimulate studies on carcinogenesisinduced by AFB1.