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目的采用逆转录病毒载体介导的RNAi技术靶向抑制乳腺癌细胞MCF-7 E2F1蛋白的表达。方法构建重组的表达E2F1siRNA的逆转录病毒载体,将重组载体和空载体转染入MCF-7细胞中,筛选出稳定转染的单克隆细胞扩大培养,应用Western Blot方法检测各组细胞E2F1蛋白的表达情况。结果筛选出4个转染逆转录病毒载体的单克隆,命名为SIR1~4,其E2F1蛋白表达量分别为0.776±0.012,0.768±0.013,0.764±0.012,0.720±0.016;转染空载体组和未转染细胞组E2F1蛋白表达量分别为1.512±0.011和1.494±0.013;SIR1~4 E2F1蛋白表达量低于空载体组及未转染组,差异有统计学意义(P<0.05),SIR1~4各组E2F1蛋白表达均受到抑制;空载体转染组与未转染组之间差异无统计学意义(P>0.05)。结论逆转录病毒载体介导的RNAi技术特异、高效抑制靶基因的表达,为肿瘤基因治疗提供了新的思路和工具。
Objective To reverse the expression of E2F1 protein in MCF-7 breast cancer cells by retroviral vector-mediated RNAi. Methods Recombinant E2F1 siRNA retroviral vector was constructed and transfected into MCF-7 cells. Stably transfected monoclonal cells were screened for expansion. Western Blot method was used to detect the expression of E2F1 protein Express the situation. Results Four monoclonal anti-retroviral vectors were selected and named as SIR1-4. The E2F1 protein expression levels were 0.776 ± 0.012, 0.768 ± 0.013, 0.764 ± 0.012 and 0.720 ± 0.016, respectively. The transfected empty vector group and The expression of E2F1 protein in untransfected cells was 1.512 ± 0.011 and 1.494 ± 0.013, respectively; the expression of SIR1 ~ 4 E2F1 protein was lower than that in empty vector and untransfected cells (P <0.05) 4 E2F1 protein expression in each group were inhibited; empty vector transfected group and non-transfected group, the difference was not statistically significant (P> 0.05). Conclusion The retroviral vector-mediated RNAi technology is specific and efficient in inhibiting the expression of target genes, providing a new idea and tool for gene therapy of tumors.