有机阴离子转运体1抑制剂筛选模型的构建及应用

发布时间：2018-05-26 19:30

本文选题：马丁达比狗肾上皮细胞 + 有机阴离子转运体　；参考：《中国药学杂志》2017年01期

【摘要】：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。
[Abstract]:Objective to establish a transgenic cell model of Madin-Darby canine kidneyne MDCK-1 cells which can stably express organic anion transporter (1(organic anion transporter OAT1) in the kidney epithelium of Martin Darby canine, and to screen the inhibitors of OAT1 by using this model. Methods the plasmid pcDNA3.1 () was transfected into MDCK cells by Lipofectamine~(TM)2000 transfection reagent by liposome transfection, and the positive clones were obtained by G418 screening. The activity of OAT1 was determined by the accumulation of 6-carboxyfluorescein (6-carboxyfluorescein 6-CFL) in monoclonal cells and in the presence of traditional Chinese medicine monomers. The mRNA expression of OAT1 was detected by real-time quantitative polymerase chain reaction (PCR) and quantitative real-tinle PCR. The uptake of 6-CFL in MDCK-mock and MDCK-OAT1 cells and the inhibition of 6-CFL uptake by traditional Chinese medicine monomer were studied by using stable cell lines. Results qRT-PCR results showed that the relative expression of OAT1 mRNA in MDCK-OAT1 cells was 4 862 times higher than that in mock cells. The ability of uptake of 6-CFL was 14.9 times of that of the latter. 6-CFL in the cell line was 42.36 渭 mol / L ~ (-1). Some traditional Chinese medicine monomers could significantly inhibit the ability of MDCK-OAT1 to transport 6-CFL. Conclusion the OAT1 inhibitor screening model was successfully constructed, and it can quickly and effectively screen out Chinese traditional medicine monomers with potential inhibitory effect on OAT1, which provides the experimental basis for the plant-drug interaction based on OAT1 in the later stage.
【作者单位】：南方医科大学药学院广东省新药筛选重点实验室;
【基金】：广东省科技计划项目(2014A020210013)
【分类号】：R94

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