膀胱癌稳定表达多药耐受相关蛋白亚克隆的生物学特征

Biological characters of a multidrug resistance related protein overexpression subline of the bladder carci-noma

目的 观察稳定表达多药耐受相关蛋白 (multidrugresistance relatedprotein ,MRP)的膀胱癌细胞有关多药耐受 (multidrugresistance ,MDR)的生物学特征。方法 将全长MRPcDNA转染膀胱癌EJ细胞 ,并建立稳定表达MRP的亚克隆EJ/MRP ,在光镜和透射电镜下观察细胞形态和生长情况 ,用蛋白质免疫印迹和流式细胞仪 (FCM)方法检测多药耐受糖蛋白 (P gp)、MRP的表达和细胞内药物聚集及滞留的情况。结果 和对照EJ/Vect细胞相比 ,EJ/MRP细胞胞浆内出现丰富的囊泡 ,MRP荧光染色率增加了 6.2倍 ,差异有显著性 (P <0 .0 5) ,但 2种细胞均无P gp表达 ;EJ/MRP细胞内的药物聚集浓度大约只有EJ/Vect细胞的 67% ,前者外排药物速度快于后者。结论 EJ/MRP稳定高表达MRP ,MRP介导的MDR表型和细胞内囊泡出现。

Objective To investigate biological characters of a multidrug resistant associated protein (MRP) overexpression subline of a bladder carcinoma cell line.Methods After transfected with the full length MRP,cDNA,a stable MRP overexpression subline named EJ/MRP was established. Cell growth and morphological characters were observed under light microscopy and transmission electron microscopy,and protein expression of MRP and multidrug resistance 1 (mdr1) gene were detected by using immunoblot and flow cytometry(FCM),and intracellular drug accumulation and efflux were also detected by FCM.Results Compared with control cell EJ/Vect (transfected with empty vector),EJ/MRP had rich vesicles in cytoplasm,and its fluorescence stain rate of MRP was increased 6.2 times which had a significant difference between EJ/MRP and EV/Vect(P<0.05).But both cell lines had no obvious P gp expression.The intracellular drug accumulation in EJ/MRP was about 67% of that in EJ/Vect,and intracellular drug efflux in EJ/MRP was much fast than that in EJ/VECT.Conclusion EJ/MRP over expressed MRP stable without P gp expression and MRP mediated MDR phenotype was related with the rich vesicles in cytoplasm,decrease of intracellular drug accumulation and increase of intracellular drug efflux.