NLS-RARα基因沉默对HL-60细胞增殖及分化的影响

Effect of NLS-RARα gene silence on proliferation and differentiation of HL-60 cells

目的探讨抑制NLS-RARα基因表达对急性早幼粒细胞白血病(Acute promyeolic leukemia,APL)细胞株HL-60增殖及分化的影响。方法将针对NLS-RARα基因的shRNA真核表达质粒(干扰组)和阴性对照质粒(阴性对照组)采用脂质体法转染HL-60细胞,并设未转染组,经G418筛选出稳定转染的细胞,采用MTT法检测细胞的增殖活力;RT-PCR和QRT-PCR法检测细胞中NLS-RARα基因mRNA的转录水平;Western blot法检测细胞中NLS-RARα蛋白的表达水平;流式细胞术检测细胞表面分化抗原CD11b的表达及细胞周期的分布。结果与阴性对照组和未转染组比较,干扰组HL-60细胞的增殖活力、NLS-RARα基因mRNA的转录水平和蛋白的表达水平均明显下降(P<0.05);CD11b的表达明显升高(P<0.05);G1、G2期细胞比例明显增加,S期细胞比例明显减少(P<0.05)。结论抑制NLS-RARα基因的表达可抑制HL-60细胞增殖,促进其分化。本实验为进一步研究APL发生发展的机制及APL的分子诊断和靶向治疗新途径奠定了基础。

Objective To explore the effect of inhibition of NLS-RARot gene expression on the proliferation and differentiation of acute promyeolic leukemia （APL） HL-60 cells. Methods HL-60 cells were transfected with the shRNA eukaryotic expression vector targeting NLS-RARα and negative control plasmid in mediation of liposome respectively, using those untransfected as normal control. The stably transfected cells were screened with G418, and determined for proliferation activity by MTY method, for transcription level of NLS-RARα mRNA by RT-PCR and QRT-PCR, for expression level of NLS-RARot protein by Western blot, and for expression of cell surface differentiation antigen CD1 lb and distribution of cell cycle by flow cytometry （FCM）. Results Compared with those in negative and normal control groups, the proliferation activity, transcription level of NLS-RARα mRNA and expression level of NLS-RARα protein HL-60 cells transfected with shRNA eukaryotic expression vector targeting NLS-RARα decreased significantly （P 〈 0. 05）, while the expression level of CD1 lb increased significantly （P 〈 0. 05 ）; however, the percentages of cells at G1 and G2 phases increased significantly, while that at S phase decreased significantly （P 〈 0. 05 ）. Conclusion The inhibition of NLS-RARct gene expression suppressed the proliferation and promoted the differentiation of HL-60 ceils, which laid a foundation of further study on mechanism of onset and development as well as molecular diagnosis and novel pathway to target therapy of APL.