【摘要】 目的探讨乳腺癌耐药蛋白(BCRP)在卵巢癌中的作用和逆转其介导的MDR,建立表达BCRP的耐药细胞系3AO/BCRP。方法提取MCF-7细胞的总RNA,设计BCRP基因上下游引物,RT-PCR克隆出BCRP基因全长并连接到pcDNA3.1(-)上,转染3AO细胞后以G418筛选存活细胞。采用米托蒽醌外排实验、细胞毒性实验、细胞群体倍增时间、定量PCR、Western blot等方法鉴定构建的耐药细胞系。结果3AO/BCRP对米托蒽醌耐药指数增加至11.58;耐药细胞外排米托蒽醌的作用增强;细胞中BCRPmRNA的水平升高,细胞能表达一定量的BCRP。结论3AO/BCRP细胞具有BCRP的耐药表型,可作为进一步研究BCRP的生物学特性以及建立其介导的耐药逆转模型。更多还原

【Abstract】 Objective To explore the function of BCRP in ovary cancer and establisment of BCRP expressing ovary cancer cell line 3AO/BCRP.Methods We construct the BCRP expressing cell line 3AO/BCRP.Extract the total RNA of MCF-7 cells,clone the whole length of BCRP gene by RT-PCR and ligate the gene to pcDNA3.1(-),transfect the positive clones to 3AO cells and select positive cell clones with G418,identify the survival cells by MTT,efflux assay,western blot.Results The multidrug resistance index of 3AO/BCRP to mitoxantrone increased to 11.58 times;the efflux of 3AO/BCRP to mitoxantrone enhanced;the relative level of BCRP mRNA in 3AO/BCRP increased and western blot indicate that the 3AO/BCRP cells can express more protein than 3AO cells.Conclusion 3AO/BCRP is a reliable BCRP expressing cell model,the establishment of this cell line has laid basis for further study of the mechanism of BCRP.更多还原