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目的探讨缺失核定位信号的PML,即PML(NLS-)干扰对急性早幼粒细胞白血病细胞HL-60增殖与凋亡的影响。方法将靶向PML(NLS-)基因的3组干扰质粒pGpu6-PML(NLS-)shRNA和阴性对照质粒pGpu6-NCshRNA分别转染HL-60细胞,转染后48 h,G418筛选阳性克隆,分别命名为Si-1、Si-2、Si-3和NC组,并设空白对照组。采用RT-PCR法和Western blot法检测各组细胞中PML(NLS-)基因mRNA的转录水平和蛋白的表达水平;MTT法检测细胞的增殖活力;流式细胞术分析细胞的细胞周期及凋亡情况。结果 Si-1和Si-2组HL-60细胞PML(NLS-)基因mRNA的转录水平和蛋白的表达水平与空白对照组相比明显减低(P<0.05),有干扰效果;Si-1组HL-60细胞的增殖水平与空白对照组相比明显降低(P<0.05),以转染后48 h降低最为显著(P<0.01);抑制PML(NLS-)表达可引起HL-60细胞S期比例增高,G1和G2期比例下降(P<0.05);Si-1组细胞凋亡率明显高于NC组和空白对照组(P<0.05)。结论干扰PML(NLS-)的表达可促进HL-60细胞的凋亡,抑制其增殖。
Objective To investigate the effect of PML lacking nuclear localization signal (PML) on the proliferation and apoptosis of acute promyelocytic leukemia HL-60 cells. Methods The three interfering plasmids pGpu6-PML (NLS-) shRNA targeting the PML (NLS-) gene and the negative control plasmid pGpu6-NCshRNA were transfected into HL-60 cells respectively. 48 h after transfection, the positive clones were screened by G418 Named Si-1, Si-2, Si-3 and NC group, and set a blank control group. The transcription level and protein expression of PML (NLS-) mRNA in each group were detected by RT-PCR and Western blot. Cell viability was measured by MTT assay. Cell cycle and apoptosis were analyzed by flow cytometry Happening. Results Compared with the blank control group, the mRNA and protein expression of PML (NLS-) gene in HL-60 cells of Si-1 and Si-2 groups were significantly decreased (P <0.05) The proliferation of HL-60 cells was significantly lower than that of the blank control group (P <0.05), and decreased most significantly at 48 h after transfection (P <0.01). Inhibition of PML (NLS-) (P <0.05). The apoptosis rate in Si-1 group was significantly higher than that in NC group and blank control group (P <0.05). Conclusion The interference of PML (NLS-) expression can promote the apoptosis of HL-60 cells and inhibit its proliferation.