稳定表达hMATE1及共表达hMATE1与hOCT1或hOCT2细胞模型的构建

Establishment of MDCK cell models expressing human MATE1 or co-expressing with human OCT1 or OCT2

为构建稳定表达人多药及毒素外排转运体1(h MATE1)的转基因细胞模型,提取人肾总m RNA,经逆转录PCR获得h MATE1 c DNA,借助HindⅢ、KpnⅠ两个酶切位点与pc DNA3.1(+)重组获得重组质粒。将pc DNA3.1(+)-h MATE1重组质粒转染至MDCK、MDCK-h OCT1和MDCK-h OCT2细胞中,经潮霉素B抗性筛选后,以4',6-二脒基-2-苯基吲哚(DAPI)和N-甲基-4-苯基吡啶(MPP+)的积聚实验筛选获得具有良好h MATE1功能的单克隆。测定筛选获得的细胞中转运体m RNA的表达量,并表征其对二甲双胍的积聚或对西咪替丁的转运能力。结果表明,本研究构建的MDCK-h MATE1、MDCK-h OCT1/h MATE1、MDCK-h OCT2/h MATE1细胞模型均高表达h MATE1 m RNA,MDCK-h MATE1细胞对二甲双胍的积聚为转染空载体细胞的17.6倍;MDCK-h OCT1/h MATE1和MDCK-h OCT2/h MATE1细胞对西咪替丁的净外排率分别为17.5和3.65。因此,本研究成功构建了稳定表达h MATE1及共表达h MATE1与h OCT1或h OCT2的细胞模型,可用于h MATE1及其与h OCT1或h OCT2共同参与的药物转运或药物-药物相互作用的体外研究。

To establish single- and double-transfected transgenic cells stably expressing h MATE1, h MATE1 c DNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pc DNA3.1（＋） plasmid by virtue of both HindⅢ and KpnⅠ restriction enzyme sites. Subsequently, the recombined pc DNA3.1（＋）-h MATE1 plasmid was transfected into MDCK, MDCK-h OCT1 or MDCK-h OCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 μg·m L-1, all clones were screened with DAPI and MPP＋ as substrates to identify the best candidate. The m RNA content of h MATE1, the cellular accumulation of metformin with or without cimetidine as inhibitor, or transportation of cimetidine was further valuated. The results showed that all of the three cell models over expressed h MATE1 m RNA. The cellular accumulation of metformin in MDCK-h MATE1 was 17.6 folds of the control cell, which was significantly inhibited by 100 μmol·L-1 cimetidine. The transcellular transport parameter net efflux ratios of cimetidine across MDCK-h OCT1/h MATE1 and MDCK-h OCT2/h MATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good h MATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving h MATE1 alone or together with h OCT1/2 in vitro.