表达BCRP的胚肾细胞系HEK293/BCRP的建立及生物学特征

目的:建立HEK293/BCRP多药耐药细胞系并研究其生物学功能。方法:构建有BCRP基因的表达载体,利用脂质体转染方法将载体转入HEK293细胞,并用RT-PCR、间接免疫荧光染色和流式细胞术分析检测转染细胞系BCRP的表达,体外细胞毒试验检测其对米托蒽醌等的耐药指数,流式细胞术和激光共聚焦分别检测其对罗丹明123和阿霉素的外排作用。结果:HEK293/BCRP细胞系在BCRP mRNA和蛋白表达均明显升高(P<0.05),对米托蒽醌的耐药指数达112.07倍,经1.5 h和3 h外排,细胞内罗丹明123浓度分别降低了42.25%和69.01%,激光共聚焦显示细胞内阿霉素浓度降低。结论:成功构建了表达BCRP的胚肾细胞系HEK293/BCRP,并对多种抗肿瘤药物具有耐受性。

Establishment of a human embryonic kidney epithelial cell line HEK293/BCRP expresses BCRP and its biological characterization

Objective:To establish a human multi-drug resistant embryonic kidney epithelial cell line HEK293/BCRP and investigate its biological characterization.Methods:The expression vector containing breast cancer resistance protein(BCRP) gene was constructed and transfected into HEK293 cells mediated by liposomes.The expression of BCRP was detected by RT-PCR,indirect immunofluorescence staining,and flow cytometry.The multidrug resistance index to mitoxantrone was measured by in vitro cytotoxicity test.Efflux of Rhodamine 123(Rh123) and adriamycin(ADR) was determined by flow cytometry and laser scanning confocal microscopy,respectively.Results:BCRP expression significantly increased at both mRNA and protein levels in HEK293/BCRP cells.The resistance index to mitoxantrone was 112.07 times.The intracellular concentrations of Rh123 decreased by 42.25% and 69.01% after 1.5-h and 3-h efflux,respectively.Laser scanning confocal microscopy showed that the concentration of ADR also decreased.Conclusion:We successfully establish HEK293/BCRP cell line highly expressed BCRP and has cross-resistance to several anti-cancer drugs.