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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 construct a transgenic cell model stably expressing human multidrug and toxin transporter 1 (h MATE1), total human RNA was extracted from human kidney and h MATE1 c DNA was obtained by reverse transcription PCR. Hind Ⅲ and Kpn Ⅰ restriction sites Recombinant plasmids were obtained by recombination with pcDNA3.1 (+). The pcDNA3.1 (+) - h MATE1 recombinant plasmid was transfected into MDCK, MDCK-h OCT1 and MDCK-h OCT2 cells. After screened by hygromycin B resistance, Accumulation experiments of 2-phenylindole (DAPI) and N-methyl-4-phenylpyridine (MPP +) were screened to obtain a monoclonal clone with good h MATE1 function. The amount of m RNA expression of the transporter in the cells obtained by the screening was determined and its accumulation of metformin or its ability to transport cimetidine was characterized. The results showed that h MATE1 m RNA was highly expressed in MDCK-h MATE1, MDCK-h OCT1 / h MATE1 and MDCK-h OCT2 / h MATE1 cell models, and the accumulation of metformin in MDCK-h MATE1 cells was transfected empty 17.6 times of vector cells. The net efflux rates of cimetidine by MDCK-h OCT1 / h MATE1 and MDCK-h OCT2 / h MATE1 cells were 17.5 and 3.65, respectively. Therefore, the present study successfully constructed a cell model stably expressing h MATE1 and co-expressing h MATE1 and h OCT1 or h OCT2, which can be used for h MATE1 and its drug transport or drug-drug interaction which is involved in h OCT1 or h OCT2 In vitro research.