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目的探讨小分子干扰RNA(smallinterferingRNAs,siRNA)对A2780/Taxol细胞多药耐药性的逆转作用。方法2005-07-2005-12华中科技大学同济医学院附属协和医院根据多药耐药基因1(MDR1)序列设计2条siRNA,将siRNA包装入质粒,经转化、克隆、扩增、纯化,与pEGFPN-1质粒一起共转染A2780/Taxol细胞,流式细胞仪、MTT、RT-PCR和Westernblot分别检测转染效率、紫杉醇的半数抑制浓度(IC50)、细胞P-gp蛋白及MDR1mRNA表达。结果两种重组质粒与EGFP质粒转染效率无明显差异,P-gp表达水平明显下调。两条siR-NA对紫杉醇敏感性的相对逆转率分别达63·8%及51·2%。MDR1基因不同程度的沉默,MDR-A能更有效封闭MDR1基因,MDR1mRNA相对水平下降(67·3±0·8)%。结论siRNA能有效逆转MDR1基因编码P-gp介导的A2780/Taxol细胞多药耐药。
Objective To investigate the reversal effects of small interfering RNAs (siRNAs) on multidrug resistance in A2780 / Taxol cells. METHODS: Two siRNAs were designed according to the multidrug resistance gene 1 (MDR1) sequence of Huazhong University of Science and Technology Affiliated Union Hospital, Huazhong University of Science and Technology. The siRNA was packaged into plasmid and transformed, cloned, amplified and purified. The A2780 / Taxol cells were cotransfected with pEGFPN-1 plasmid. The transfection efficiency, the IC50 of paclitaxel, the expression of P-gp protein and MDR1 mRNA were detected by flow cytometry, MTT, RT-PCR and Western blot respectively. Results The transfection efficiencies of the two recombinant plasmids and EGFP plasmids were not significantly different, and the expression of P-gp was significantly down-regulated. The relative reversal rates of the two siR-NA to paclitaxel were 63.8% and 51.2% respectively. MDR1 gene silenced to some extent, MDR-A could block MDR1 gene more effectively, and the relative level of MDR1 mRNA decreased by 67.3 ± 0.8%. Conclusion siRNA can effectively reverse MDR1 gene encoding P-gp mediated A2780 / Taxol multidrug resistance.