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应用Tet-On基因表达系统,调控CYP2E1基因在NIH3T3细胞中的表达水平,探讨CYP2E1在化学致癌物二甲基亚硝胺(N-nitrosodimethylamine,NDMA)代谢中的作用.先后将Tet-on基因表达系统的调控质粒pRevTet-on和反应质粒pRevTRE-2E1转染NIH3T3细胞,分别用G418和潮霉素筛选,并通过RT-PCR和Western印迹检测,成功获得了3个具有良好诱导性Tet调控的CYP2E1基因表达细胞系(Tet/3T3-2E1).应用不同浓度强力霉素(doxycycline,Dox)处理Tet/3T3-2E1细胞诱导CYP2E1表达,HPLC分析细胞对CYP2E1特异性药物探针氯唑沙腙的原位代谢能力及MTT法分析CYP2E1介导的NDMA细胞毒性作用.结果显示,Tet/3T3-2E1细胞CYP2E1的表达及其代谢能力有明显的Dox浓度依赖性,而且NDMA对Dox诱导组细胞有明显浓度依赖性细胞毒性作用,其IC50值为12.06μmol/L,无Dox诱导组未见明显的NDMA细胞毒性作用.该细胞模型的成功建立对于开展与CYP2E1相关的毒物、致癌物代谢研究,筛选抗毒物、抗癌物等具有重要价值.
Tet-On gene expression system was used to regulate the expression of CYP2E1 in NIH3T3 cells and to explore the role of CYP2E1 in the metabolism of chemical carcinogens N-nitrosodimethylamine (NDMA). Tet-on gene expression Systemic control plasmid pRevTet-on and the reaction plasmid pRevTRE-2E1 were transfected into NIH3T3 cells and screened by G418 and hygromycin respectively. Three CYP2E1s with good induced Tet regulation were obtained successfully by RT-PCR and Western blotting. (Tet / 3T3-2E1) .TET / 3T3-2E1 cells were treated with different concentrations of doxycycline (Dox) to induce the expression of CYP2E1, and the cytotoxicity of CYP2E1-specific drug probe chlorazolone Metabolism and MTT assay were used to analyze CYP2E1-mediated NDMA cytotoxicity.The results showed that the expression of CYP2E1 in Tet / 3T3-2E1 cells and its metabolic capacity were significantly Dox concentration-dependent, and NDMA Dox-induced cells were significantly concentration Dependent cytotoxicity with an IC50 value of 12.06 μmol / L and no significant NDMA cytotoxicity was observed in the non-Dox-induced group.The successful establishment of this cell model was of great value in the development of CYP2E1-related toxicants, carcinogen metabolism Research, screening anti-drugs, anti-cancer substances have important value.