P-糖蛋白高表达马丁达比狗肾上皮细胞系的建立  

Establishment of Madin-Darby Canine Kidney Cell Line with High P-Glycoprotein Expression

作　　者：刘瑶[1] 俞春娜[1] 曾苏[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》2009年第21期1608-1613,共6页Chinese Pharmaceutical Journal

基　　金：国家"十一五"重大科研专项;临床前药物代谢动力学技术平台(2009ZX09304-003)

摘　　要：目的为建立能稳定表达人P-糖蛋白(P-glycoprotein,P-gp)的马丁达比狗肾上皮(Madin-Darby canine kidney,MDCK)细胞转基因细胞系,并鉴定其是否适用于药物转运实验。方法将质粒pcDNA3.1(+)/MDR1通过LipofectamineTM2000转染试剂转染犬肾上皮细胞MDCK,经G418筛选获得抗性克隆。通过P-gp的荧光底物罗丹明(Rhodamine123,Rho123)在各单克隆细胞内的积聚量以及维拉帕米存在时细胞内积聚量检测P-gp的活性。反转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)和蛋白免疫印迹(Westernblot)检测人源多药耐药基因1(multidrug resistance1,MDR1)的mRNA和P-gp表达量。应用筛选出的MDCK-MDR1单克隆细胞株,研究了Rho123在MDCK和MDCK-MDR1(Madin-Darby canine kidney/multidrug resistance1)细胞中的转运及4种经典抑制剂(维拉帕米、环孢素、酮康唑和奎宁)对Rho123转运的抑制特性。结果RT-PCR和Western blot检测表明,所建立的MDCK-MDR1细胞与MDCK细胞相比,经转染后MDCK细胞内的MDR1的信使核糖核酸(messenger ribonucleic acid,mRNA)和P-gp表达发生从无到有的变化。Rho123在MDCK和MDCK-MDR1中的外排系数分别是5.94和15.45,抑制剂能显著地抑制细胞外排作用。结论转染后的细胞高表达P-gp,适用于研究药物的肠道和血脑屏障转运实验。OBJECTIVE To establish Madin-Darby canine kidney （MDCK） cell line with stable expression of human P-glycoprotein （P-gp） and scree an effective model to study drug transportation. METHODS The plasmid pcDNA3.1 （＋）/MDR1 was transfected into MDCK cell line using LipofectamineTM 2000 transfection reagent. Several stable transfected clones were obtained after selection with G418. Rhodamine123 （Rho123）, the fluorescence probes substrate, was used to study the profiles of effiux and uptake to test P-gp activities, which was performed with MDCK and MDCK-MDR1 （Madin-Darby canine kidney/multidrug resistance 1）cells in a one-chamber system with the presence or absence of verapamil as inhibitor. The expression of P-gp messenger ribonueleic acid （mRNA） and protein was detected by using reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. This study measured the bidirectional transport of Rho123 across MDCK and MDCK-MDRI monolayers with the absence or presence of four well-known P-gp inhibitors （verapamil, cyclosporina, ketoeonazole, quinine）. RESULTS The RT-PCR and Western blot result indicated high homo sapiens P-gp expression in transfected cells comparing with controls. The Rho123 effiux ratios of MDCK to MDCK-MDR1 were 5.94 and 15.45, respectively. Several known P-gp inhibitors could significantly reduced the polarized effiux. CONCLUSION The developed MDCK-MDR1 cell line is an effective model to study drug transportation in body.

关 键 词：马丁达比狗肾上皮细胞 多药耐药基因1 P-糖蛋白 转运 外排 

分 类 号：R915[医药卫生—微生物与生化药学]