短发夹RNA干扰表达质粒逆转人乳腺癌细胞MCF-7/ADR多药耐药的研究

目的构建靶向多药耐药基因（MDR-1）的短发夹RNA（short hairpin loop RNA shRNA）干扰表达质粒，并探讨其逆转人乳腺癌细胞MCF-7/ADR多药耐药的作用效果。方法根据MDR-1基因表达序列设计有效的RNA干扰片断，构建并获得MDR-1基因特异的shRNA质粒表达载体pRNAT-U6.1/Neo－mdr1，采用lipofectin2000分别转染人乳腺癌耐药细胞MCF-7/ADR，利用G418筛选稳定表达的细胞克隆。利用RT-PCR检测MDR-1-RNA的表达，Western Blotting检测MDR-1蛋白质的表达，MTT法检测耐药逆转效果，罗丹明123外排实验检测p-gp的转运功能。结果成功构建表达siRNA的质粒载体pRNAT-U6.1/Neo－mdr1，转染人乳腺癌耐药细胞MCF-7/ADR后48h及G418筛选后1、2月，mdr1-RNA及蛋白质表达均呈明显下降，p-gp的转运功能明显提高，对阿霉素的敏感性增强，与耐药细胞及转入空白载体的细胞比较，差异有统计学意义（P均＜0.05）。筛选后1、2月与转入48h比较，差异无统计学意义（P＞0.05）。结论shRNA干扰表达质粒pRNAT-U6.1/Neo-mdr1能够稳定、持久的抑制MDR-1基因，逆转乳腺癌细胞MCF-7/ADR对阿霉素的耐药性。

Reversal of multi-drug resistance in human breast cancer MCF-7/ADR cells by short hairpin RNA expression plasmids

Objective To construct shRNA plasmids which target the MDR1 gene and to investigate their reversal effect on the human breast cancer cell line MCF-7/ADR.Methods Oligonucleotides of the MDR-1 gene were designed based on the Gene Bank and the MDR-1 shRNA expression plasmid pRNAT-U6.1/Neo-mdr1 was constructed.The human breast cancer cell line MCF-7/ADR was tranfected with the MDR-1 shRNA expression plasmids by lipofectamine 2000,and a positive clone was selected by G418.MDR-1mRNA was assayed by RT-PCR and protein expression was determined by Western blotting.The P-gp function was determined by rhodamine 123 retention and the efficiency of MCF-7/ADR to ADM was determined by the MTT method.Results MDR-1 shRNA expression plasmids were successfully constructed.The MDR-1mRNA and protein expressions were significantly decreased 48 hours after transfection,1 month and 2 months after selection by G418,and sensitivity to P-gp-transportable drugs was restored.The transporting function of P-gp was increased and the efficiency of MCF-7/ADR to ADM significantly reversed. Conclusions The shRNA expression plasmid pRNAT-U6.1/Neo-mdr1 can permanently inhibit MDR-1 gene stability.The sensitivity of MCF-7/ADR to adriamycin is reversed.