CYP2A13/MRP2双表达Flp-In^TM CHO细胞系的建立

Establishment of Flp-In^IM CHO cell lines with double expression of CYP2A13 and MRP2

目的构建稳定表达细胞色素P450家族2亚家族A成员13(CYP2A13)和多药耐药相关蛋白2(MRP2)的双转染Flp-In^TM CHO细胞系.方法分别构建pCMV6-NEO-CYP2A13和pcDNA5-MRP2重组质粒.先将pCMV6-NEO-CYP2A13重组质粒转染至Flp-In^TM CHO细胞中,通过有限稀释法和4-甲基亚硝胺-1-3-呲啶基-1-丁酮(NNK)细胞毒性实验筛选CYP2A13活性较高的CYP2A13-Flp-In^TMCHO细胞.再将pcDNA5-MRP2转染至CYP2A13-Flp-In^TMCHO细胞中.用实时定量P眈CR、Western blot法和NNK细胞毒性实验,检测双转染细胞及正常细胞中CYP2AI3和MRP2的表达量及其活性,筛选稳定表达CYP2A13和MRP2的FlpInTMCHO细胞.结果相较于未转染细胞,CYP2AI3-Flp-In^TMCHO细胞的CYP2A13表达增加,NNK毒性敏感度增加;CYP2A13/MRP2-Flp-In^TM CHO细胞的CYP2A13和MRP2表达也明显增加.和CYP2AI3-Flp-In^TM CHO细胞相比,CYP2A13/MRP2-Flp-In^TM CHO细胞的CYP2A13表达量无明显差异,MRP2表达增加,NNK毒性敏感度明显降低.结论成功建立了CYP2A13和MR凹的双转染细胞模型,为呼吸道致癌物质原位激活研究奠定了基础.

Objective To construct a double transfected Flp-In^TM CHO cell line stably expressing both cytochrome P450 family 2 subfamily A member 13(CYP2A13)and multidrug resistance-associated protein 2(MRP2).Methods We constructed the recombinant plasmids of pCMV6-NEO-CYP2A13 and pcDNA5-MRP2.The pCMV6-NEO-CYP2A13 recombinant plasmid was first transfected into Flp-In^TM CHO cells,and CYP2A13-Flp-ln^TM CHO cells with higher CYP2A13 activity were screened using limiting dilution method and 4-(methylnitrosamino)-1-(3-pyridyl)-l-butanone(NNK)cytotoxicity assay.Thereafter,pcDNA5-MRP2 was transfected into CYP2A13-Flp-ln^TM CHO cells.The expression levels and activities of CYP2A13 and MRP2 in the double transfected cells and normal cells were detected by real-time quantitative PCR,Western blot analysis and NNK cytotoxicity assay in order to screen Flp-In^TM GHO cells with stable expression of CYP2A13 and MRP2.Results Compared with non-transfected cells,the expression of CYP2A13 and the sensitivity of NNK toxicity in CYP2A13-Flp-ln^TM CHO cells increased.The expression of CYP2A13 and MRP2 in CYP2A13/MRP2-Flp-ln^TM CHO cells also increased significantly.Compared with CYP2A13-Flp-ln^TM CHO cells,CYP2A13/MRP2-FIp-ln^TM CHO cells showed no significant difference in OYP2A13 expression;the expression of MRP2 increased while the sensitivity of NNK toxicity decreased significantly.Conclusion The double transfected cell model of CYP2A13 and MRP2 has been successfully established,which lays the foundation for the study of in situ activation of respiratory carcinogens.