[Establishment of MDCK cell models expressing human MATE1 or co-expressing with human OCT1 or OCT2].
[Establishment of MDCK cell models expressing human MATE1 or co-expressing with human OCT1 or OCT2].
复制标题
DOI：
--
复制
发表时间：
2015-07
期刊：
影响因子：
--
通讯作者：
H. Lei;Siwen Sun;Li-ping Li-Li-ping-Li-1651122038;Mei-juan Tu;Hui Zhou;S. Zeng;Huidi Jiang
中科院分区：
文献类型：
--
作者：
H. Lei;Siwen Sun;Li-ping Li-Li-ping-Li-1651122038;Mei-juan Tu;Hui Zhou;S. Zeng;Huidi Jiang
为建立稳定表达hMATE1的单、双价转基因细胞，采用RT-PCR方法从冻存的人肾组织中克隆hMATE1基因，并通过HindIII和Kpn I酶切位点将其亚克隆到pcDNA3.1(+)载体中。用脂质体2000试剂将重组的真核表达载体pcDNA3.1(+)-hMATE1分别导入MDCK、MDCK-hOCT1和MDCK-hOCT2细胞。用400µg·mL(-1)的潮霉素B培养14d后，以DAPI和MPP+为底物对所有克隆进行筛选，筛选出最佳菌株。进一步观察hMATE1mRNA含量、西咪替丁抑制前后二甲双胍的细胞蓄积及西咪替丁转运情况。结果表明，三种细胞模型均过度表达hMATE1mRNA。100umol.L(-1)西咪替丁可显著抑制二甲双胍在MDCK-hMATE1细胞中的蓄积量，为对照细胞的17.6倍。西咪替丁在MDCK-hOCT1/hMATE1和MDCK-hOCT2/hMATE1单层的跨细胞转运参数净外排比分别为17.5和3.65。综上所述，成功建立了hMATE1功能良好的细胞模型，可用于研究hMATE1单独作用或与hOCT1/2共同作用下的药物转运或药物相互作用。
To establish single- and double-transfected transgenic cells stably expressing hMATE1, hMATE1 cDNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pcDNA3.1(+) plasmid by virtue of both HindIII and Kpn I restriction enzyme sites. Subsequently, the recombined pcDNA3.1(+)- hMATE1 plasmid was transfected into MDCK, MDCK-hOCT1 or MDCK-hOCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 µg · mL(-1), all clones were screened with DAPI and MPP+ as substrates to identify the best candidate. The mRNA content of hMATE1, 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 hMATE1 mRNA. The cellular accumulation of metformin in MDCK-hMATE1 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-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good hMATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving hMATE1 alone or together with hOCT1/2 in vitro.