稳定表达人OCTN1/OCTN2的细胞模型构建及应用  

Establishment and application of cell models with stable expression of hOCTN1/2

作　　者：翁娅韵 金李莎 汪宇清[1] 宋飞凤[1] 李丽萍[1] 周慧[1] 曾苏[1] 蒋惠娣[1] 

机构地区：[1]浙江大学药学院药物代谢与分析实验室,浙江省抗肿瘤药物重点实验室,浙江杭州310058 [2]浙江工业大学长三角绿色制药协同创新中心,浙江杭州310014

出　　处：《药学学报》2016年第6期931-937,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81373474);浙江省科技厅资助项目(2015C33162)

摘　　要：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 transporter 1 and 2（hOCTN1 and hOCTN2） mediate transport of endogenous and exogenous compounds. The present study aimed to establish cell models with stable expression of hOCTN1 or hOCTN2 to study interactions with compounds and transporters. MDCK cells were transfected with pcDNA3.1（＋） plasmid vector containing hOCTN1 or hOCTN2（pcDNA3.1（＋）-hOCTN1/2）, several stable transfected clones were obtained after G418 screening. hOCTN1 and hOCTN2 clones were screened with ergothioneine and mildronate respectively as substrates to identify the best candidates. We explored interactions of endogenous substances, alkaloids, flavonoids and ACEIs with hOCTN1/2. As a result, the cellular accumulation of ergothioneine in MDCK-hOCTN1 or mildronate in MDCK-hOCTN2 was 122 and 108 folds of the control cells, respectively. The kinetic parameters, K_m and V_（max） of ergothioneine, mediated by MDCK-hOCTN1, were 8.19 ± 0.61 μmol·L^（-1） and 1 427 ± 49 pmol·mg^（-1）（protein）·min^（-1）; while K_m and V_（max） of mildronate by MDCK- hOCTN2 were 52.3 ± 4.3 μmol·L^（-1） and 2 454 ± 64 pmol·mg^（-1）（protein）·min^（-1）. Dopamine, glutamine, piperine, berberine, nuciferine, lisinopril and fosinopril could inhibit ergothioneine or mildronate uptake by MDCK- hOCTN1/2. In conclusion, cell models with good stable hOCTN1 and hOCTN2 functions have been established successfully, which can be applied to the study of interactions between compounds and transporters of hOCTN1 and hOCTN2.

关 键 词：人肉碱/有机阳离子转运体1和2 麦角硫因 米屈肼 抑制作用 犬肾上皮细胞 

分 类 号：R943[医药卫生—药剂学]