人源有机阴离子转运多肽1B1和多药耐药相关蛋白2双转MDCKⅡ细胞株的构建及其功能验证  

Establishment of double-transfected MDCKⅡ cells expressing human organic anion transporting polypeptide 1B1 and multidrug resistance-associated protein 2 and identification of its functions

作　　者：胡楠[1] 马国[2] 杨青[1,3] 

机构地区：[1]复旦大学生命科学学院生物化学系,上海200438 [2]复旦大学药学院临床药学教研室,上海201203 [3]云南天然产物与生物制药协同创新中心,昆明650091

出　　处：《复旦学报（医学版）》2017年第2期134-142,共9页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金面上项目(81573310;81374051);高等学校博士学科点专项科研基金(20130071110037)~~

摘　　要：目的构建人源有机阴离子转运多肽1B1(human organic anion transporting polypeptide 1B1,hOATP1B1)和多药耐药相关蛋白2(multidrug resistance-associated protein2,hMRP2)双转MDCKⅡ细胞株,验证其功能,并应用其考察创新药物2,3-双加氧化酶(indoleamine 2,3-dioxygenase,IDO)抑制剂1-甲基色氨酸(1-methyl-tryptophan,1-MT)的转运特性。方法采用基因工程手段获得hOATP1B1和hMRP2表达真核载体pVITRO2-SLCO1B1-ABCC2,转染MDCKⅡ细胞,通过遗传霉素G418筛选得到稳定表达的细胞株;通过Realtime PCR、Western blot、免疫荧光共聚焦显微镜确认目的蛋白特异性;利用该双转模型考察普伐他汀(不同pH环境和不同底物浓度)和1-MT的转运。结果经电泳分析、双酶切、DNA测序鉴定表明重组质粒构建成功;通过Real-time PCR、Western blot、免疫荧光共聚焦显微镜证明经遗传霉素筛选的MDCK-OATP1B1/MRP2细胞构建成功;pH=6.5时,普伐他汀在所构建双转细胞模型上转运最佳;在0~500μmol/L的浓度范围内,普伐他汀在该模型上的转运呈现浓度依赖性。1-MT在该细胞模型上无明显转运。结论成功构建人源MDCKOATP1B1/MRP2双转细胞株,发现1-MT既不是OATP1B1蛋白也不是MRP2蛋白的底物,该细胞株可用于OATP1B1/MRP2介导的外源性物质(如药物)和内源性物质(如胆红素)的转运研究。Objective To establish double-transfected Madin-Darby canine kidney （MDCK） Ⅱ cells expressing human organic anion transporting polypeptide 1B1 （hOATP1B1） and multidrug resistance-associated protein 2 （hMRP2） and to testify their functions, moreover, to study the transcellur transport of indoleamine 2,3-dioxygenase （IDO） inhibitor 1-methyltryptophan （1-MT） in the transfectants. Methods hOATP1B1/hMRP2 eukaryotic vectors pVITRO2-SLCO1B1-ABCC2 was obtained by genetic engineering method and then transfected into MDCK cells. Stably expressed MDCK cells were screened by using the geneticin G418. Real-time PCR, Western blot analysis and immuno fluorescent confocal microscopy were used to verify the proteins expression. Transport of the representative substrate pravastatin in different pH values and substrate concentrations and 1-MT were evaluated using the double transfectants. Results MDCK-OATP1B1/MRP2 was successfully established. Pravastatin displayed the optimal transcellular transport when pH value was 6.5. Transport of pravastatin demonstrated the concentration-dependent in the concertation range of 0 to 500 μmol/L. Transport of 1-MT showed no significant difference in MDCK cells and transfectants. Conclusions MDCK-OATP1B1/MRP2 was successful established; 1-MT was not the substrate of OATP1B1 or MRP2 protein; and the eatablished double transfectant cell lines can be used to evaluate OATP1B1/MRP2-medicated transport of xenobiotics （e.g. new drug candidates） and endogenous compounds （e.g. bilirubin）.

关 键 词：有机阴离子转运多肽1B1 多药耐药相关蛋白2 MDCKⅡ 普伐他汀 1-甲基色氨酸 转运 

分 类 号：R965.1[医药卫生—药理学] Q233[医药卫生—药学]