【摘要】 目的探讨靶向增强型绿色荧光蛋白(EGFP)小发夹结构RNA(ShorthairpinRNA,shRNA)对耐阿霉素的人乳腺癌细胞(MCF-7/AdrR)中EGFP基因表达的抑制作用。方法构建针对EGFP基因的shRNA表达载体,与pcDNA3.0EGFP质粒共转染MCF-7/AdrR细胞,分别用激光共聚焦扫描显微镜(Laserconfocalscanningmicroscope,LCSM)和流式细胞术检测干扰效果。结果瞬时共转染pSilencerTM3.1-H1neoEGFPshRNA质粒和pcDNA3.0EGFP质粒后,荧光显微镜观察结果显示MCF-7/AdrR细胞中发出荧光的细胞数量减少,荧光强度明显降低。流式细胞术检测阳性细胞百分率降低至35.6%,差异有显著性。结论靶向EGFP的shRNA表达质粒能有效而特异抑制EGFP在MCF-7/AdrR中的表达,MCF-7/AdrR细胞中存在RNA干扰现象,为进一步利用RNA干扰技术逆转乳腺癌多药耐药性的研究奠定基础。更多还原

【Abstract】 Objective:To investigate whether short hairpin RNA expression vector targeting enhanced green fluo in multidrug resistance human breast cancer cells(MCF-7/AdrR).Methods:The shRNA expression vector targeting EGFP gene was constructed, and co-transfected into MCF-7 cells and MCF-7/AdrR cells with pcDNA3.0 EGFP. The RNAi effects were assessed by laser confocal scanning microscope and flow cytometry.Results:In MCF-7/AdrR cells transient co-transfected with pSilencer TM 3.1-H1 neo shRNA and pcDNA3.0 EGFP expression vectors,LCSM showed that positive percentage of EGFP expression was reduced,and fluorescence intensity was depressed significantly.FCM assay showed that positive percentage was significantly reduced to 35.6%(P<0.05).Conclusion:shRNA expression vector targeting EGFP gene could inhibit enhanced green fluorescent protein expression in MCF-7/AdrR cells effectively and specifically, RNA interference exists in MCF-7/AdrR cell line, which will provide a new strategy for researching the reversal of multidrug resistance by RNAi in breast cancer cells.更多还原