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构建MDCK-hMDR1-CYP3A4双转表达体系,研究CYP3A4和P-gp之间的相互作用.根据基因文库合成CYP3A4基因编码片段,连接到pcDNA3.1/Hypgro表达载体,进行测序.验证正确的重组质粒pcDNA3.1/Hypgro/hCYP3 A4转染MDCK-MOCK细胞以及MDCK-hMDR1细胞,经潮霉素B(HygromycinB)加压筛选阳性克隆,获得稳定高表达人CYP3 A4的MDCK-hCYP3A4单转以及MDCK-hMDR1-hCYP3A4双转稳转细胞株.结果显示相比于空白细胞(MDCK-MOCK)以及阴性细胞(MDCK-MDR1),CYP3A4在MDCK-hCYP3A4、MDCK-hMDR1-hCYP3A4细胞中的表达更稳定并且活性显著增强.MTT结果表明,在MOCK、MDCK-MDR1、MDCK-CYP3A4以及MDCK-MDR1-CYP3A4 4种细胞系中,苏尼替尼的IC50值分别为2.832,5.717,3.628,6.561μ,mol/L,而伯舒替尼的IC50值分别为0.712 6,4.413,2.184,7.010μmol/L.由于苏尼替尼和博苏替尼是CYP3A4以及P-gp的共同底物,CYP3 A4的代谢活性以及P-gp的外排功能共同作用使得苏尼替尼和博苏替尼毒性降低,因而MDCK-hMDR1-hCYP3A4双转细胞株IC50值比MDCK-hMDR1、MDCK-hCYP3 A4明显增大.以上结果说明了构建的稳定单转MDCK-hCYP3A4以及MDCK-hMDR1-hCYP3A4双转细胞株是具有功能的,并且CYP3A4与P-gp之间的作用是相互影响的,同时该模型的建立也为药物的转运与代谢研究提供更全面的研究技术与思路.“,”P-glycoprotein(P-gp) and cytochrome P450 3A4 enzymes are known to play an important role in oral bioavailabilities drugs.The present study is aiming at investigating the interplay between CYP3A4 and P-gp through constructing MDCK-hMDR1-hCYP3A4 double transfected cell line.Synthesizing CYP3A4 coding region gene from human gene bank and cloning it into pcDNA3.1/Hypgro plasmid,and then verifying gene sequence accuracy by sequence analysis.The recombinant vector containing hCYP3A4 cDNA was transfected into MDCK-MOCK cells and MDCK-hMDR1 cells using LipofectAMINE 2000 reagent,and the single cell colonies stable expressing hCYP3A4 were picked out after the transfected cells culturod in complete medium containing.Hygromycin B (0.3 mg/mL) to obtain single-transfected cell line MDCK-hCYP3A4 and double-transfected cell line MDCK-hMDR1-hCP3A4.The expression of CYP3A4 in MDCK-MOCK,MDCK-hCYP3A4,MDCK-hMDR1 and MDCK-hMDR1-hCP3A4 cell lines was detected by RT-PCR and Western Blotting.The enzyme activity of CYP3A4 in these four cell lines was identified by the metabolism assay of the probe substrate CYP3A4.The results indicated that the expression of CYP3A4 in MDCK-hCYP3A4 and MDCK-hMDR1-hCP3A4 cell lines were much higher than the MOCK cell and the negative cell (MDCK-hMDR1).Furthermore,1-Hydroxymidazolam,the metabolife of midazolam (CYP 3A4 substrate),were generated much more in MDCK-hCYP3A4 and MDCK-hMDR1-hCP3A4 cell lines compared with the MOCK cell and the negative cell (MDCK-hMDR1).MrTr assay results showed that the IC50 of sunitinib in MOCK,MDCK-hMDR1,MDCK-hCYP3A4,MDCK-MDR1-CYP3A4 cell lines was 2.832,5.717,3.628,6.561 μmol/L,respectively.And the IC50 of bosutinib were 0.712 6,4.413,2.184,7.010 μmol/L in MOCK,MDCK-hMDR1,MDCK-hCYP3A4,MDCK-MDR1-CYP3A4 cell lines,respectively.Due to the metabolism by CYP3A4 or P-gp efflux,the toxicity of sunitinib or bosutinib in MDCK-hMDR1 or MDCK-hCYP3A4 cell lines was less than MOCK cells.And in MDCK-hMDR1-hCYP3A4 cell line,the toxicity decreased dramatically because of the interplay between CYP3A4 and P-gp.Therefore,the MDCK-hCYP3A4 and MDCK-hMDR1-hCYP3A4 cell lines were functional and could be applied to study more comprehensively in drug metabolism and transport.