稳定表达人有机阳离子转运体1的细胞模型构建  

Establishment of Cell Model with Stable Expression of Organic Cation Transporter 1

作　　者：涂美娟[1] 陈忠坚[1] 孙思源[1] 石爱琴[1] 李丽萍[1] 曾苏[1] 蒋惠娣[1] 

机构地区：[1]浙江大学药学院药物分析与药物代谢研究室,杭州310058

出　　处：《中国药学杂志》2011年第20期1546-1551,共6页Chinese Pharmaceutical Journal

基　　金："十一五"重大新药创制专项资助(2009ZX09304-003);国家自然科学基金资助(81072689)

摘　　要：目的建立能稳定表达人有机阳离子转运体1(human organic cation transporter1,hOCT1)野生型及两个突变体的马丁达比狗肾上皮(Madin-Darby canine kidney,MDCK)细胞模型。方法从人肝组织中提取hOCT1野生型基因,经定点突变获得hOCT1P341L,hOCT1M420del两个突变型基因,构建表达质粒pcDNA3.1(+)-hOCT1,pcDNA3.1(+)-hOCT1P341L,pcDNA3.1(+)-hOCT1M420del。将质粒转染MDCK细胞,通过G418筛选获得抗性克隆,并通过hOCT1的荧光底物(4-二甲氨基苯乙烯基)-N-甲基吡啶(ASP+)筛选得到具有较高活性的MDCK-hOCT1细胞。经反转录聚合酶链式反应(RT-PCR)及经典底物N-甲基-4-苯基吡啶(MPP+)的摄取实验,鉴定筛选得到的单克隆细胞株hOCT1 mRNA的表达及功能。结果本实验获得的hOCT1野生型及2个突变体细胞模型与转染空载体的MDCK细胞相比,其hOCT1 mRNA表达量显著增高,对经典底物MPP+的摄取为转染空载体细胞的12.5,13.7,12.0倍,hOCT1抑制剂可显著抑制细胞对MPP+的摄取。结论建立的细胞模型可用于hOCT1抑制剂及底物的筛选。OBJECTIVE To establish Madin-Darby canine kidney（MDCK） cell model with stable-expressed wild type human organic cation transporter 1（hOCT1） and its two mutants.METHODS The hOCT1 wild-type gene was extracted from human liver,and the two mutants,hOCT1P341L,and hOCT1M420del,were obtained by site-directed mutagenesis.The plasmids pcDNA3.1（＋）-hOCT1,pcDNA3.1（＋）-hOCT1P341L and pcDNA3.1（＋）-hOCT1M420del were constructed and transfected into MDCK cell line.Several stable transfected clones were obtained by G418 screening.The activity of hOCT1 was estimated by detecting the amount of hOCT1 fluorescent substrate,4-（4-di-methylaminostyryl）-N-methyl-pyridinium（ASP＋）,accumulation with or without inhibitor tetraethylammonium（TEA＋） in clones.The mRNA of hOCT1 was detected by reverse transcription-polymerase chain reaction（RT-PCR）.The function of selected effective monoclones was confirmed by evaluating the accumulation of hOCT1 classical substrate,1-methyl-4-phenylpyridinium（MPP＋）.RESULTS The RT-PCR results showed high hOCT1 mRNA expression in the selected cells compared with MDCK empty vector cells.The accumulation amount of MPP＋ in the developed MDCK-hOCT1,hOCT1P341L and hOCT1M420del cells were 12.5,13.7 and 12.0 times of those of MDCK empty vector cells,and the inhibitor TEA＋ could significantly reduce the accumulation.CONCLUSION The developed MDCK-hOCT1,hOCT1P341L and hOCT1M420del cells are effective in vitro models for screening hOCT1 substrates and inhibitors.

关 键 词：人有机阳离子转运体1 马丁达比狗肾上皮细胞 基因多态性 细胞模型 

分 类 号：R915[医药卫生—微生物与生化药学]