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目的建立能稳定表达有机阴离子转运体1(organic anion transporter,OAT1)的马丁达比狗肾上皮(Madin-Darby canine kidney,MDCK)细胞转基因细胞模型,利用该模型初步筛选OAT1的抑制剂。方法采用脂质体转染法将质粒pcDNA3.1(+)通过Lipofectamine~(TM)2000转染试剂转染MDCK细胞,并经G418筛选获得阳性克隆。通过OAT1的经典底物6-羧基荧光素(6-carboxyfluorescein,6-CFL)在单克隆细胞内的积聚量以及中药单体存在时细胞内的积聚量来检测OAT1的活性。实时荧光定量聚合酶链反应(quantitative real-tinle PCR)检测OAT1的mRNA的表达量。利用稳定细胞株,研究了6-CFL在MDCK-mock和MDCK-OAT1细胞中的摄取、中药单体对6-CFL摄取的抑制特性。结果 qRT-PCR结果表明,所建立的MDCK-OAT1细胞与mock细胞相比,OAT1 mRNA相对表达量为后者的4 862倍,摄取6-CFL的能力为后者的14.9倍。6-CFL在细胞株中摄取的K_m值为42.36μmol·L~(-1),部分中药单体能显著抑制MDCK-OAT1转运6-CFL的能力。结论 OAT1抑制剂筛选模型构建成功,并能快速有效地筛选出对OAT1有潜在抑制作用的中药单体,为后期的基于OAT1的植物-药物相互作用提供实验基础。
OBJECTIVE: To establish a transgenic mouse model of Madin-Darby canine kidney (MDCK) stably expressing the organic anion transporter (OAT1), and to screen the inhibitor of OAT1 by this model. Methods The plasmid pcDNA3.1 (+) was transfected into MDCK cells by LipofectamineTM 2000 transfection reagent. The positive clones were screened by G418. The activity of OAT1 was detected by the accumulation of 6-carboxyfluorescein (6-CFL), a classical substrate of OAT1, in the monoclonal cell and the amount of intracellular accumulation in the presence of Chinese traditional medicine. Real-time quantitative polymerase chain reaction (quantitative real-tinle PCR) OAT1 mRNA expression was detected. The uptake of 6-CFL in MDCK-mock and MDCK-OAT1 cells and the inhibition of 6-CFL uptake by Chinese herbs were studied using stable cell lines. Results qRT-PCR results showed that compared with mock cells, the relative expression level of OAT1 mRNA in MDCK-OAT1 cells was 4 862-fold and 14.9-fold higher than that in 6-CFL. The K_m value of 6-CFL uptake in cell lines was 42.36μmol·L -1, and some Chinese herbs could significantly inhibit the ability of MDCK-OAT1 to transport 6-CFL. Conclusion The OAT1 inhibitor screening model is successfully constructed and can rapidly and effectively screen out the Chinese traditional medicine monomer that has the potential inhibitory effect on OAT1 and provide experimental basis for later OAT1-based plant-drug interaction.