外源化合物对孕烷X受体和组成型雄烷受体介导的细胞色素P4503A4和2B6转录调节共转染体系的优化  

Optimization of cotransfection system for hPXR and hCAR mediated transcriptional activation of CYP3A4 and CYP2B6 by xenobiotics

作　　者：关志男[1] 王燕[1] 王锐利[1] 张丽锋[1] 张淑秋[1] 

机构地区：[1]山西医科大学药学院,太原030001

出　　处：《中国药物与临床》2012年第6期696-699,共4页Chinese Remedies & Clinics

基　　金：国家自然科学基金(30973589);山西省基础研究项目(2009021040-1);山西省留学人员科研资助项目(2009-47)

摘　　要：目的优化孕烷X受体(hPXR)和组成型雄烷受体(hCAR)介导的细胞色素P450(CPY)3A4和CYP2B6诱导共转染体系,提高检测系统的灵敏度。方法利用invitrogen脂质体2000共同转染表达质粒hPXR/hCAR、报告基因质粒CPY3A4/CYP2B6和内参质粒pRL-TK到HepG-2细胞中。系统以hPXR的激动剂利福平,hCAR的激动剂CITCO为阳性对照组,以二甲基亚砜(DMSO)为溶剂阴性对照组。通过调整3种质粒的转染比例,以利福平/DMSO和CITCO/DMSO的比活值,即阳性药物的诱导倍数作为优化系统灵敏度的指标,分别获得最大比值以表示系统具有最佳灵敏度。结果当共转染体系比例为hPXR/hCAR表达质粒150ng、CPY3A4/CYP2B6报告基因质粒600ng、PLR-TK内参质50ng时,转染体系的检测灵敏度最高。结论针对所使用的转染细胞系和共转染质粒,通过优化质粒的转染比例可提高系统的灵敏度,优化的共转染系统可用于药物代谢酶诱导机制的研究。Objective To optimize cotransfection system of hPXR and hCAR mediated transcriptional activation to CYP3A4 and CYP2B6, and to improve the sensitivity of the detection system. Methods Liposome 2000 （Invitrogen） was used to cotransfect expression plasmid hPXR/hCAR, reporter gene plasmid CPY3A4/CYP2B6 and reference plasmid pRL-TK into HepG-2 cells. Rifampicin, agonist of hPXP, and CITCO, agonist of hCAR, were taken for positive solvent controls; DMSO was used as negative control. The proportion of the three cotransfeetion plasmids was adjusted, and the activity ratios of rifampiein to DMSO and CITCO to DMSO, i.e. the induction multiples of positive solvents, were used as the index for evaluating the optimized system sensitivity. Those with the biggest ratios implied the system had optimized sensitivity. Results The system showed the highest sensitivity when the proportion was at hPXR/hCAR 150 rig, CYP3A4/CYP2B6 600 ng and pRL-TK 50 rig. Conclusion Higher sensitivity may be obtained by optimizing the proportion of cotransfection plasmids. The optimized eotransfeetion system can be used to study the induction mechanism of drug metabolizing enzymes in vitro.

关 键 词：细胞色素P450系统 转染 人孕烷X受体 人组成型雄烷受体 

分 类 号：R96[医药卫生—药理学]