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目的利用短发夹RNA(shorthairpinRNA,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(multidrugresistance,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,Westernblotting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pSilencerTM3.1-H1neoMDR1-A和MDR1-BshRNA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%,Westernblotting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MDR1shRNA表达质粒增加MCF-7/AdrR细胞内柔红霉素的积累。MDR1shRNA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 the multidrug resistance (MDR) of doxorubicin-resistant human breast cancer cell line (MCF-7 / AdrR) by short hairpin RNA (shRNA) expression vector. Methods Two MDR1 gene shRNA expression plasmids were constructed and stably transfected into MCF-7 / AdrR cells. The expression of MDR1 gene mRNA was detected by RT-PCR, the expression of P-glycoprotein (P-gp) was detected by Western blotting, the apoptosis and the sensitivity to doxorubicin were detected by flow cytometry and MTT respectively. Focus fluorescence microscopy to detect intracellular daunorubicin steady-state accumulation. Results The mRNA expression of MDR1 in MCF-7 / AdrR cells stably transfected with pSilencerTM3.1-H1neoMDR1-A and MDR1-BshRNA plasmids was reduced to 37.6% and 28.0% respectively by RT-PCR. Western blotting results showed that P- gp expression was significantly and specifically inhibited. The resistance of transfected cells to doxorubicin was reduced from 162 fold to 108 fold and 50 fold respectively, and MDR1 shRNA expression plasmid increased the daunorubicin accumulation in MCF-7 / AdrR cells. MDR1 shRNA expression plasmid combined with doxorubicin can induce apoptosis in MCF-7 / AdrR cells. Conclusion The shRNA expression plasmid effectively reverses the multidrug resistance and renders the drug-resistant tumor cells regain the sensitivity to chemotherapeutic drugs.