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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 a cell model of Madin-Darby canine kidney (MDCK) stably expressing wild type and two mutants of human organic cation transporter1 (hOCT1). Methods Wild type hOCT1 gene was extracted from human liver tissue and two mutant genes of hOCT1P341L and hOCT1M420del were obtained by site - directed mutagenesis. The expression plasmids of pcDNA3.1 (+) - hOCT1, pcDNA3.1 (+) - hOCT1P341L and pcDNA3.1 +) - hOCT1M420del. The plasmids were transfected into MDCK cells. The resistant clones were screened by G418 and screened by the fluorescent substrate (4-dimethylaminostyryl) -N-methylpyridine (ASP +) of hOCT1 to obtain MDCK- hOCT1 cells. The hOCT1 mRNA expression and function of the selected monoclonal cell lines were identified by reverse transcription-polymerase chain reaction (RT-PCR) and classical substrate N-methyl-4-phenylpyridine (MPP +) uptake assay. Results Compared with MDCK cells transfected with empty vector, the expression levels of hOCT1 mRNA in hOCT1 wild type and two mutant cell lines were significantly increased. Uptake of classical substrate MPP + into transfected empty vector cells was 12.5, 13.7,12.0 times, hOCT1 inhibitors can significantly inhibit the cell uptake of MPP +. Conclusion The established cell model can be used for hOCT1 inhibitors and screening of substrates.