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LipofectamineTM 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-time 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Km值为42.36 μmol·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 cell model stably expressing mouse organic anion transporter1(OAT1) in MDCK cells, for the purpose of screening potent OAT1 inhibitors in vitro. METHODS Recombinant plasmid pcDNA3.1(+)-OAT1 was constructed and transfected into MDCK cells using LipofectamineTM 2000 reagent. After the process of G418 screening, cells were collected for further validation. Cells were harvested, and the quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was carried out to test the OAT1 mRNA expression in MDCK-OAT1 cells. The function of the stably transfected cells were validated by the uptake activity of (6-Carboxyfluorescein,6-CFL ),a substrate of OAT1.The inhibitors of OAT1 were selected according to their inhibition activity towards the uptake of 6-CFL into the OAT1-over expressing cells in comparison with the typical inhibitor of OAT1, probenecid. RESULTS The pcDNA3.1(+)-OAT1 was well conducted. The mRNA expression of OAT1 was significantly higher than that in mock cells; MDCK-OAT1 cells had a significantly high mRNA expression comparing with the mock cells, being 4 862 fold of that in mock cells. The uptake-ability of 6-CFL in MDCK-OAT1 and MDCK-mock cells was obviously different, with a 14.9 fold increase in comparison with mock cells. In the presence of probenecid and several monomers from Chinese herbs, fluorescence values in cell lysates were reduced to varying degrees, and results showed that rhein, luteolin, chrysin and quercetin could significantly inhibited the 6-CFL uptake mediated by hOAT1,with a reduction of more than 80% of the control. CONCLUSION The aim to establish a cell model which could stably express OAT1 is achieved. Further study could be done using this cell model, for the screening of potential inhibitors of OAT1 from monomers of Chinese herbs, and then could be used as a tool in the research of herb-drug interaction.
关键词
马丁达比狗肾上皮细胞 /
有机阴离子转运体1 /
6-羧基荧光素 /
摄取 /
中药单体
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Key words
MDCK cells /
organic anion transporter1(OAT1) /
6-CFL /
uptake /
monomers of Chinese herbs
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中图分类号：
R965
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参考文献
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脚注
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基金
广东省科技计划项目(2014A020210013)
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