药学学报, 2015, 50(7): 842-847
引用本文:
雷红梅, 孙思源, 李丽萍, 涂美娟, 周慧, 曾苏, 蒋惠娣. 稳定表达hMATE1及共表达hMATE1与hOCT1或hOCT2细胞模型的构建[J]. 药学学报, 2015, 50(7): 842-847.
LEI Hong-mei, SUN Si-yuan, LI Li-ping, TU Mei-juan, ZHOU Hui, ZENG Su, JIANG Hui-di. Establishment of MDCK cell models expressing human MATE1 or co-expressing with human OCT1 or OCT2[J]. Acta Pharmaceutica Sinica, 2015, 50(7): 842-847.

稳定表达hMATE1及共表达hMATE1与hOCT1或hOCT2细胞模型的构建
雷红梅, 孙思源, 李丽萍, 涂美娟, 周慧, 曾苏, 蒋惠娣
浙江大学药学院, 浙江 杭州 310058
摘要:
为构建稳定表达人多药及毒素外排转运体1 (hMATE1) 的转基因细胞模型, 提取人肾总mRNA, 经逆转录PCR获得hMATE1 cDNA, 借助HindⅢ、KpnⅠ两个酶切位点与pcDNA3.1(+) 重组获得重组质粒。将pcDNA3.1(+)-hMATE1重组质粒转染至MDCK、MDCK-hOCT1和MDCK-hOCT2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hMATE1功能的单克隆。测定筛选获得的细胞中转运体mRNA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-hMATE1、MDCK-hOCT1/hMATE1、MDCK-hOCT2/hMATE1细胞模型均高表达hMATE1 mRNA, MDCK-hMATE1细胞对二甲双胍的积聚为转染空载体细胞的17.6倍; MDCK-hOCT1/hMATE1和MDCK-hOCT2/hMATE1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hMATE1及共表达hMATE1与hOCT1或hOCT2的细胞模型, 可用于hMATE1及其与hOCT1或hOCT2共同参与的药物转运或药物-药物相互作用的体外研究。
关键词:    细胞模型      人多药及毒素外排转运体1      人有机阳离子转运体1      人有机阳离子转运体2      共表达     
Establishment of MDCK cell models expressing human MATE1 or co-expressing with human OCT1 or OCT2
LEI Hong-mei, SUN Si-yuan, LI Li-ping, TU Mei-juan, ZHOU Hui, ZENG Su, JIANG Hui-di
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
Abstract:
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 KpnⅠ 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.
Key words:    cell model    human multidrug and toxin extrusion 1    human organic cation transporter 1    human organic cation transporter 2    coexpression   
收稿日期: 2014-11-28
基金项目: 国家自然科学基金资助项目 (81373474).
通讯作者: 蒋惠娣
Email: hdjiang@zju.edu.cn
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