人BCRP高效稳定表达COS-7细胞系的建立及其药物外排活性鉴定  

Establishment of the high-stably expressing human BCRP-COS-7 cell line and measurement of its efflux function

作　　者：张辉[1] 贾英[1] 王珍玉 邓继峰[1] 王海峰[1] 戴仁科[1] 黄黎珍[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006 [2]中山珐玛斯医药科技有限公司,广东中山528400

出　　处：《广东药学院学报》2014年第4期493-496,共4页Academic Journal of Guangdong College of Pharmacy

基　　金：国家自然基金(81202585);中央高校基本科研业务费专项资金(2014ZM0067)

摘　　要：目的建立乳腺癌耐药蛋白BCRP高效稳定表达COS-7细胞系,为相关药物的药物转运及其耐药性研究奠定基础。方法采用RT-PCR技术从MCF-7/ADR乳腺癌细胞中扩增出乳腺癌耐药蛋白基因BCRP的cDNA序列,插入真核表达载体pcDNA3.1(+),获重组质粒pcDNA3.1(+)-BCRP,将重组质粒pcDNA3.1(+)-BCRP转染COS-7细胞,经G418筛选后获得抗性细胞克隆。运用基因组PCR、SDSPAGE鉴定抗性细胞中外源BCRP基因的整合及表达,并以BCRP底物mitoxantrone对鉴定阳性的细胞克隆进行转运活性验证。结果成功克隆BCRP的cDNA并构建真核细胞表达载体pcDNA3.1(+)-BCRP。转染COS-7细胞后获得抗性克隆10个,PCR鉴定阳性有4个,其中2个克隆能高效表达BCRP蛋白。经活性验证,2个高表达的细胞克隆中有一个能显著高效外排BCRP底物mitoxantrone,显示良好的生物活性功能。结论成功建立一株BCRP高效稳定表达的COS-7细胞系,且具有特异性生物活性,可用于BCRP相关药物转运及耐药性研究。Objective To establish the human BCRP-COS-7 cell line and provide the platform for the study of BCRP-mediated drug transport and resistance. Methods cDNA of BCRP was cloned from MCF-7/ADR cell line and constructed into pcDNA3.1（＋） . Then recombinant vector was transfected into COS-7 cells. The resulting colonies treated with G418 were selected,and the expression of BCRP was identified by genome PCR and SDS-PAGE. The transport activity of high-stably expressing human BCRP was confirmed by efflux of BCRP substrate mitoxantrone. Results The expression vector pcDNA3. 1 （＋）-BCRP was successfully constructed. COS-7 cells were transfected with pcDNA3. 1 （＋）-BCRP and ten G418-resistant colonies were selected. PCR and SDS-PAGE were used to confirm that four colonies are positive and two of them were positive expression, respectively. One colony displayed high efficiency to efflux mitoxantrone with the comparison of wild type COS-7 cells. Conclusion The high-stably expressing BCRP-COS-7 cell line has been successfully established. The BCRP efflux function to mitoxantrone is confirmed. The resulting cell line could be applied to further study of BCRP-mediated drug transport and resistance.

关 键 词：乳腺癌耐药蛋白 药物转运 COS-7 

分 类 号：R346[医药卫生—基础医学]