Tet调控CYP2E1基因表达细胞系的代谢能力分析  

Effects on Metabolism and Cytotoxicity of N-nitrosodimethylamine in a Tet-Controlled CYP2E1 Expressing Cell Line

作　　者：侯德富[1,2] 万恂恂[1] 贺智敏[2] 陈主初[2] 

机构地区：[1]湖南师范大学医学院生物化学与分子生物学教研室,长沙410006 [2]中南大学湘雅医学院肿瘤研究所,长沙410078

出　　处：《中国生物化学与分子生物学报》2009年第2期163-168,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：湖南省教育厅资助科研项目(No.08C569);湖南师范大学青年基金资助项目(No.29040625)~~

摘　　要：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.An eukaryotic Tet-on expression system was used to express CYP2E1 in NIH 3T3 cells under quantitative controls. The human CYP2E1 eDNA from nasopharynx was subcloned into the Tet-on vector （pRevTRE） and transfected into NIH 3T3 cells with the constitutively expression of a reverse tetracyclinecontrolled transactivator （rtTA）. The expression of CYP2E1 was tightly regulated in a dose-dependent manner by doxycycline （Dox）, and so was the catalytic activity of CYP2E1 as indicated by the generation of 6- hydroxychlorzoxazone （6-OH- CZ） from chlorzoxazone （ CZ）. The cytotoxicity induced by 0. 001 - 10 mmol/L N-nitrosodimethylamine （NDMA） for 4 days in Tet/3T3- CYP2E1 cells was also assayed, a Dox dose- and time-dependent decrease of cell viability was observed the IC50 of 12.06 μmol/L, whereas in the absence of Dox, a good cell viability to NDMA was observed at a concentrations up to 10 mmol/L. This cell line will be useful to assess the metabolic impact of CYP2E1, especially to investigate the role of CYP2E1 in the metabolic activation of carcinogenic nitrosamines in vitro.

关 键 词：CYP 2E1 Tet—on基因表达系统 二甲基亚硝胺 强力霉素 

分 类 号：R739.63[医药卫生—肿瘤]