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目的:探讨AKT2基因在肿瘤细胞对紫杉醇敏感性中的作用。方法:分别构建2个针对同一AKT2基因不同位点的短发卡状RNA(shRNA)载体,转染人卵巢癌细胞A2780、SKOV3,荧光显微镜观察细胞内荧光蛋白的表达及转染效率,运用RT-PCR、蛋白质免疫印迹(western blotting)方法比较单独转染的抑制瘤和共转染对AKT2表达的沉默效率;抑制AKT2表达后,流式细胞仪(FACS)检测肿瘤细胞对紫杉醇的敏感性。结果:构建的2种shRNA表达载体均可抑制AKT2 mRNA和蛋白的表达,共转染的抑制率明显高于分别单独转染的抑制率(P<0.05);FACS结果显示,共转染沉默AKT2基因后,细胞凋亡率明显增加(P<0.05)。结论:共转染针对AKT2基因不同位点的2个shRNA真核表达载体,对AKT2基因的抑制效率高于单独转染1个载体;抑制卵巢癌细胞中AKT2基因表达,能增强其对紫杉醇的敏感性。
Objective: To investigate the role of AKT2 gene in paclitaxel sensitivity in tumor cells. Methods: Two short hairpin RNA (shRNA) vectors targeting different sites of the same AKT2 gene were constructed and transfected into human ovarian cancer cells A2780 and SKOV3 respectively. The expression of fluorescent protein and transfection efficiency were observed by fluorescence microscopy. PCR and Western blotting were used to compare the silencing efficacies of AKT2 and transfected tumor suppressor and co-transfection. Flow cytometry (FACS) was used to detect the sensitivity of tumor cells to paclitaxel after inhibition of AKT2 expression. Results: The expression of AKT2 mRNA and protein were both inhibited by the constructed shRNA expression vector. The inhibition rate of co-transfection was significantly higher than that of the transfection alone (P <0.05). FACS analysis showed that co-transfection of AKT2 After the gene, the apoptosis rate was significantly increased (P <0.05). CONCLUSION: Co-transfection of two shRNA eukaryotic expression vectors targeting different sites of AKT2 gene is more efficient than AKT2 gene alone transfected into a vector. Inhibition of AKT2 gene expression in ovarian cancer cells can enhance its inhibitory effect on paclitaxel Sensitivity.