利用Tet-On系统敲低PRDM5对BEAS-2B细胞增殖的影响

Effect of PRDM5 knockdown on proliferation of BEAS-2B cells by Tet-On controlled system

[目的]探讨利用dox诱导的Tet-On调控系统敲低PRDM5基因对人正常支气管上皮细胞BEAS-2B增殖的影响。[方法]构建敲低PRDM5基因的pLKO-Tet-On-PRDM5-shRNA慢病毒质粒,包装生产慢病毒并转染BEAS-2B细胞,以相同条件制备转染pLKO-Tet-On-control-shRNA的BEAS-2B细胞作为对照。WesternBlot法检测PRDM5蛋白的表达水平;MTT实验观察细胞增殖情况;平板克隆形成实验检测细胞增殖能力;流式细胞术检测细胞凋亡水平。[结果]在合适剂量的dox诱导下(0.5μg/mL),敲低组细胞中PRDM5蛋白表达明显低于对照组(P<0.05);各时间点OD值高于对照组(P<0.05);克隆形成数大于对照组(P<0.01);凋亡率低于对照组(P<0.01)。[结论]成功构建出Tet-On系统调控敲低PRDM5的BEAS-2B细胞系。PRDM5基因敲低可促进BEAS-2B细胞增殖并且抑制其凋亡。

[Objective]To investigate the impact of knockdown of PRDM5 gene on proliferation and apoptosis of normal human bronchial epithelial cells BEAS-2B by dox induced Tet-On controlled system.[Methods] PRDM5 stably knockdown BEAS-2B cell line was generated by cotransfection of recombinant lentiviral vector containing pLKO-Tet-On-PRDM5-shRNA or the pLKO-Tet-On-control-shRNA together with packaging plasmids,respectively.The change of PRDM5 protein level was detected by Western Blot.The MTT assay was used to assess the cell proliferation ability.Colony formation assay was performed and the colony numbers were calculated.Cell apoptosis rates were detected by flow cytometry.[Results]Proper dose of dox(0.5 μg /mL)significantly decreased the protein expression of PRDM5(P<0.05).The OD values at each time point were higher than those of the control group(P<0.05).The colony formation number was higher than that of the control group(P<0.01).The apoptosis rate was lower compared to the control group(P<0.01).[Conclusion]The stable BEAS-2B cell lines with PRDM5 knockdown by Tet-On controlled system was successfully constructed.Knockdown of PRDM5 promoted cell proliferation of BEAS-2B cells and repressed the cell apoptosis in BEAS-2B cells.