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人肉碱/有机阳离子转运体1和2(human carnitine/organic cation transporters,hOCTN1/hOCTN2)参与多种内、外源物质转运。本研究拟构建稳定表达hOCTN1/2的细胞模型,用于药物与转运体相互作用的研究。将重组质粒pcDNA3.1(+)-hOCTN1/2转染MDCK细胞,经G418抗性筛选挑取单克隆细胞株,以麦角硫因(OCTN1经典底物)或米屈肼(OCTN2经典底物)进行细胞积聚研究,挑选功能最优的MDCK-hOCTN1/2细胞株为模型细胞。进一步考察经典底物的积聚动力学,并探究内源物、生物碱、黄酮及普利类药物对hOCTN1/2的抑制作用。结果显示,麦角硫因在MDCK-hOCTN1细胞上的积聚量为mock细胞的122倍,K_m和V_(max)为8.19±0.61μmol·L~(-1)和1 427±49 pmol·mg~(-1)(protein)·min~(-1);米屈肼在MDCK-hOCTN2细胞上的积聚量为阴性对照(mock细胞)的108倍,K_m和V_(max)为52.3±4.3μmol·L~(-1)和2 454±64 pmol·mg~(-1)(protein)·min~(-1)。多巴胺、谷氨酰胺、胡椒碱、黄连素、荷叶碱、赖诺普利和福辛普利对hOCTN1/2有显著的抑制作用。因此,本研究构建的MDCK-hOCTN1/2细胞模型可应用于药物与hOCTN1/hOCTN2相互作用研究。
Human carnitine / organic cation transporters 1 and 2 (hOCTN1 / hOCTN2) are involved in the transport of many internal and foreign substances. This study was to construct a cell model stably expressing hOCTN1 / 2, which was used to study the interaction between drug and transporter. The recombinant plasmid pcDNA3.1 (+) - hOCTN1 / 2 was transfected into MDCK cells. The monoclonal cell lines were selected by G418 resistance screening. The cells were treated with ergothioneine (OCTN1 classic substrate) or Meldonium (OCTN2 classic substrate) Cell accumulation studies were performed to select the best-performing MDCK-hOCTN1 / 2 cell lines as model cells. Further investigate the accumulation kinetics of classical substrates, and explore the inhibition of hOCTN1 / 2 by endogenous, alkaloid, flavonoids and Pulix drugs. The results showed that the accumulation of ergothionein in MDCK-hOCTN1 cells was 122-fold higher than that of mock cells with K_m and Vmax of 8.19 ± 0.61μmol·L -1 and 4227 ± 49 pmol · mg ~ (-1) -1) (protein) · min ~ (-1). The amount of mometralose accumulated in MDCK-hOCTN2 cells was 108-fold higher than that of mock cells, with Km and Vmax of 52.3 ± 4.3μmol·L ~ (-1) and 2 454 ± 64 pmol · mg ~ (-1) protein · min ~ (-1), respectively. Dopamine, glutamine, piperine, berberine, lotus leaf, lisinopril and fosinopril had a significant inhibitory effect on hOCTN1 / 2. Therefore, the MDCK-hOCTN1 / 2 cell model constructed in this study can be applied to the study of drug interactions with hOCTN1 / hOCTN2.