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目的 建立人细胞色素P4 5 0 2E1(CYP2E1)cDNA体外异源表达模型 ,观察化学致癌物二亚硝基哌嗪 (N ,N’ dinitrosopiperazine ,DNP)对CYPWE1表达的影响。方法 脂质体介导转染法将外源人CYP2E1cDNA导入NIH3T3细胞 ,PCR和Southernblot鉴定外源基因的整合 ,RT PCR检测其表达 ;用不同浓度乙醇和DNP处理转化细胞 ,RT PCR和westernbloting检测CYP2E1表达的改变。结果 获得二个具外源基因CYP2E1cDNA整合及稳定表达的细胞克隆NIH3T3 2E1 A1和NIH3T3 2E1 A8;经乙醇和DNP处理的细胞克隆CYP2E1mRNA和蛋白质水平的表达增强。结论 DNP与乙醇对CYP2E1表达的诱导可能与其mRNA转录增强有关 ,DNP致癌可能与CYP2E1对其原位代谢活化相关
OBJECTIVE: To establish a heterologous expression pattern of human cytochrome P450 02E1 (CYP2E1) in vitro and to investigate the effect of DNP on CYPWE1 expression. METHODS: Foreign human CYP2E1 cDNA was transfected into NIH3T3 cells by lipofectamine. The integration of exogenous genes was confirmed by PCR and Southern blot. The expression of CYP2E1 was detected by RT PCR. The transformed cells were treated with different concentrations of ethanol and DNP. The expression of CYP2E1 Changes in expression. RESULTS: Two cell clones, NIH3T3 2E1 A1 and NIH3T3 2E1 A8, which had the CYP2E1 cDNA integration and stable expression were obtained. The expression of CYP2E1 mRNA and protein in cell clones treated with ethanol and DNP was enhanced. Conclusions The induction of CYP2E1 expression by DNP and ethanol may be related to the increase of its mRNA transcription. The carcinogenesis of DNP may be related to the in situ metabolism of CYP2E1