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150 ng、CPY3A4/CYP2B6报告基因质粒600 ng、PLR-TK内参质50 ng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 trans crip tionaj activation to CYP3A4 and CYP2B6, and to improve the sensitivity of the detection system. Methods Liposome 2000 (In-vitrogen) was used to cotransfect expression plasmid hPXR/hCAR, reporter gene plasmid CPY3A4/GYP2B6 and reference plasmid pRL-TK into HepG-2 cells. Rifampicin, agonist of hPXP, and C1TCO, agonist of hCAR, were taken for positive solvent controls; DMSO was used as negative control. The proportion of the three cotransfection plasmids was adjusted, and the activity ratios of rifampicin 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 ng, CYP3A4/CYP2B6 600 ng and pRL-TK 50 ng. Conclusion Higher sensitivity may be obtained by optimizing the proportion of cotransfeetion plasmids. The optimized cotransfeetion system can be used to study the induction mechanism of drug metabolizing enzymes in, vitro.
展开▼
