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.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
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)
晓庄学院青年基金资助项目(No.29040625)~~
