摘要
目的:建立稳定表达乳腺癌耐药蛋白(Breast cancer resistance protein,BCRP)的小鼠成纤维细胞系PA317/BCRP,初步探讨其耐药机制。方法:从乳腺癌耐药细胞系MCF-7/ADR中克隆BCRP基因。将编码序列克隆导入真核表达载体pcDNA3.1,转化感受态E.Coli DH5α,获得重组质粒pcDNA3.1/BCRP,酶切并测序鉴定。用电穿孔法将质粒转染PA317细胞,同时转染pcDNA3.1/EGFP。G418筛选阳性克隆。阳性转染细胞提取细胞总RNA,RT-PCR检测BCRP的mRNA表达,Western blot检测BCRP蛋白表达。MTT法检测耐药克隆PA317/BCRP细胞对米托蒽醌(Mitoxantrone,MTZ)耐药性,罗丹明123外排实验验证转染后细胞外排罗丹明123功能。Western blot检测PI3K-AKT信号转导通路下游靶点AKT表达变化。结果:构建质粒pcDNA3.1/BCRP,转染PA317细胞,G418筛选。RT-PCR和Western blot,均检测到细胞中BCRP基因转录及蛋白表达。与空质粒转染细胞、未转染细胞对照组相比,PA317/BCRP细胞对MTZ耐药性增强,且外排罗丹明123能力增强。检测PI3K-AKT途径下游靶点AKT蛋白磷酸化水平在PA317/BCRP细胞内明显增高。结论:1、成功获得稳定表达BCRP的PA317细胞系—PA317/BCRP,经MTF和罗丹明123外排实验证实,其耐药和外排功能增强。2、检测PA317/BCRP细胞PI3K-AKT途径下游靶点AKT的表达,观察到耐药细胞的AKT表达含量明显高于转染空质粒和未转染的PA317细胞。推测BCRP可能通过影响AKT表达上调发挥其耐药作用。
Objective： To establish a cell line expressing BCRP and investigate the mechanism of its drug resistance. Methods： The aimed segments were isolated from the cell line Mcf-7/ADR by reverse transcription polymerase chain reaction and were inserted into a eukaryotic expression plasmid pcDNA3.1 to construct a recombinant expression plasmid pcDNA3.1/BCRP. The recombinant plasmid was first propagated in Escherichia coil DH5α, then was extracted and purified and digested with BamH Ⅰ and Xho Ⅰ and was confirmed to contain full length BCRP cDNA by agarose gel analysis and DNA sequence analysis. Then the PA317 cells were transfected with the plasmid using electroporation. The rate of transfection was observed in the cells with the plasmid pcDNA3.1/EGFP. Then the transfected cells were screened with antibiotic G418. Single clones expressing BCRP were obtained through limited-dilution method. The mRNA expression of BCRP in positive clones was detected by RT-PCR. The expression of BCRP protein in positive clones was evaluated by Western blot. When we got the drug-resistance cell line, we did the cell venenosus trial with MTZ and excretion of Rho123 trial to explore the BCRP protein function. We detected the Akt protein of PI3K-Akt pathway to investigate the mechanism of its drug resistance. Results： A recombinant eukaryotic expression plasmid PCDNA3.1-BCRP was successfully established. Then the mixed PA317 clones expressing BCRP were selected by G418 for two weeks after transfection. Through limited-dilution cloning, three single clones were ob-tained, and the best one was named as Bl1. The BCRP mRNA transcription was detected by RT-PCR in the positive clones. The BCRP protein was identified from PA317/BCRP by Western blot. The cell venenosus trial with MTZ showed that the cell PA317/BCRP had the lower inhibition ratio than the cell PA317 and PA317/pcD- NA3.1. The excretion of Rho123 trial showed that the PA317/BCRP had higher excretion. Thus we proved that the cell line was stably established. Akt protein was highly expressed in PA317/BCRP, compared with that in PA317 and PA317/pcDNA3.1. Conclusion： A cell line stably expressing the BCRP protein can be established, providing a useful tool to investigate the role of BCRP in the multidrug resistance. The activation of PI3K-Akt pathway is related to multidrug resistance.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2009年第13期768-771,776,共5页
Chinese Journal of Clinical Oncology
基金
国家自然科学基金资助(编号:30670802)~~
