目的： 利用短发夹RNA（short hairpin RNA，shRNA）表达载体逆转耐阿霉素人乳腺癌细胞株(MCF-7/AdrR)的多药耐药性（multidrug resistance，MDR）。方法： 构建2个MDR1基因shRNA表达质粒，稳定转染MCF-7/AdrR细胞。RT PCR分析MDR1基因mRNA的表达，Western blotting检测P 糖蛋白(P-gp)的表达，流式细胞术和MTT法分别检测乳腺癌细胞的凋亡和对阿霉素的敏感性，激光共聚焦荧光显微镜检测细胞内柔红霉素的稳态积累。结果： 稳定转染pSilencerTM 3.1-H1 neo MDR1-A和MDR1-B shRNA表达质粒的MCF-7/AdrR细胞，RT-PCR结果显示MDR1基因mRNA表达分别减少到37.6%和280%，Western blotting结果显示P-gp表达被明显而特异地抑制。转染细胞对阿霉素的耐药性由162倍分别减低到108倍和50倍，并且MDR1 shRNA表达质粒增加MCF-7/AdrR细胞内柔红霉素的积累。MDR1 shRNA表达质粒和阿霉素联合应用可诱导MCF-7/AdrR细胞的凋亡。 结论： shRNA表达质粒有效地逆转多药耐药，使耐药的肿瘤细胞恢复对化疗药物的敏感性。

Objective: To reverse multidrug resistance (MDR) of human breast cancer cell line (MCF-7/AdrR) to adriamycin (ADM) with short hairpin RNA (shRNA) expression vectors. Methods: Two shRNA expression vectors harboring MDR1 gene were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR and P-gp expression was determined by Western blotting. The apoptosis and sensibility of the breast cancer cells to ADM were evaluated by flow cytometry and MTT assays, respectively. Cellular daunorubicin accumulation was assayed by laser scanning confocal microscope (LSCM).Results: RT-PCR showed that MDR1 mRNA expression was significantly reduced to 37.6 % (P<0.05) and 28.0% (P<0.01) in MCF-7/AdrA cells stably transfected with pSilencerTM 3.1-H1 neo MDR1-A and MDR1-B shRNA, respectively. Western blotting showed that P-gp expression was inhibited significantly and specifically. Resistance against ADM was decreased from 162-fold to 108-fold (P<0.05 ) and 50-fold (P<0.01 ). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. MDR1 shRNA vectors combined with ADM significantly induced the apoptosis of MCF-7/AdrR cells. Conclusion: shRNA vectors can effectively reverse MDR and can restore the sensitivity of drug-resistance cancer cells to conventional chemotherapeutic agents.

国家自然科学基金资助项目(No.30171064)；吉林大学创新基金资助(No.2003CX045)