【摘要】 目的构建IGF1R基因的shRNA慢病毒载体,转染A549细胞鉴定沉默效率,观察其对A549细胞增殖能力的影响。方法设计IGF1R干扰序列,与pGC-LV慢病毒载体重组形成shRNA表达载体,包装shRNA慢病毒颗粒,感染A549细胞,应用RT-PCR和Western blot检测IGF1R干扰效果;细胞生长抑制实验、克隆形成实验及细胞周期检测评价其对A549细胞增殖的影响。结果成功构建了IGF1R-shRNA重组慢病毒并高效感染A549细胞,IGF1R mRNA及蛋白表达量分别下降74.51%,70.53%;细胞群体倍增时间显著延长,克隆形成能力显著降低,细胞周期阻滞于G0/G1期。结论本研究构建的重组慢病毒能显著抑制IGF1R表达,抑制A549细胞增殖。更多还原

【Abstract】 Objective To construct the shRNA lentiviral vector targeting IGF1R gene,and to detect the efficiency of gene silence and evaluate its effect on proliferation of A549 cells.Methods DNA oligo was cloned into pGC-LV lentiviral vector to design the IGF1R siRNA targets using short hairpin.ShRNA lentivirus was harvested by virus packaging system.The interference effect of IGF1R on A549 cells was detected by RT-PCR and Western blot.A549 cell proliferation was assessed by cell growth inhibition assay,colony formation assay and cell cycle analysis.Results The IGF1R-shRNA lentiviral vector was successfully constructed.It infected A549 cells with a very high efficiency.IGF1R expression in A549 cells was decreased by 74.51%,70.53% at both mRNA and protein levels.The doubling time of cellular populations was longer,and the colony formation ability was decreased obviously.The cell was blocked at the G0/G1 phase.Conclusion The lentiviral recombinant with shRNA targeting IGF1R gene is successfully constructed and packaged,and can effectively down-regulate the IGF1R expression and inhibit the proliferation of A549 cells.更多还原