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。
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 muhidrug 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.
出处
《基础医学与临床》
CSCD
北大核心
2006年第1期61-65,共5页
Basic and Clinical Medicine
基金
国家自然科学基金(30300124)
高等学校博士学科点专项基金(20020422042)
