核干细胞因子特异性短发夹状RNA对HL-60细胞分化抗原和生物学性质的影响

Effect of specific short hairpin RNA of nucleostemin on differentiation antigens and biological properties of HL-60 cells

目的研究核干细胞因子(nuc leostem in,NS)特异性短发夹状RNA(shRNA)阻断NS基因表达对HL-60白血病细胞分化抗原及生物学性质的影响,探讨NS的生物学功能和与急性白血病的关系以及用于基因治疗的可能性。方法特异性转染试剂和合成的NS短发夹状RNA(NS-shRNA)体外直接转染HL-60细胞,采用RT-PCR判断基因阻断效果,MTT法测定转染后细胞增殖能力,流式细胞仪(FCM)测定细胞分化抗原,形态学观察细胞形状和生长状态,血细胞分析仪测定细胞大小、粒度。结果合成的2条NS-shRNA均能明显阻断NS基因表达,抑制率分别为37.82%和71.88%;NS-shRNA能显著抑制HL-60细胞的增殖并存在时间和浓度依赖性,以48 h和10 nmol/L浓度最佳。转染后:分化抗原CD11b、CD33、CD14、CD64、HLA-DR增高,CD38降低,显示有向粒系继续成熟和向单核系重新分化趋势;细胞聚团性减弱,碎片增多,一部分细胞形态变成梭形和长尾形,染色后细胞核紧密,凹陷,髓过氧化物酶(MPO)和α-醋酸萘酚酯酶(α-NAE)活性增强;大小和粒度分析小体积细胞和核碎裂细胞增多。结论NS-shRNA通过阻断NS基因表达,对HL-60细胞有抑制增殖和促分化作用,改变了细胞的生物学性质,使细胞恶性程度有所减弱,并有可能诱发凋亡,在白血病的基因中具有潜在的临床价值。

Objective To study the role of the specific short hairpin RNA （NS-shRNA） of nucleostemin in inhibiting the expression of nucleostemin （NS） gene and evaluate the effect of NS-shRNA on differentiation antigen and biological properties of HL-60 cells, so as to explore the relationship between the biological functions of NS and acute leukemia, the potential perspective of NS gene therapy with RNA interference （RNAi）. Methods HL-60 cells were directly transfected NS-shRNA by using special transfection reagent. After 48 h, the inhibition rate of NS-shRNA to NS gene was detected by RT-PCR, the proliferation ability of HL-60 cells was detected by MTT, the differentiation antigens were assayed by flow cytometry （FCM）, the morphologic peculiarities such as cell shape, growth condition were observed, and the volume and granularity of HL-60 cells were analyzed by blood cell analyzer. Results The expression of NS gene were significantly inhibited by both two NS-shRNA, the inhibition rate were 37.82% and 71.88% , respectively. The significant inhibition effect of NS-shRNA to the proliferation of HL-60 cells existed in a time-dependent and concentration-dependent manner, and the best time was 48 h, the best concentration was 10 nmol/L. The change of differentiation antigen included increase of CDllb, CD33, CD14, CD64, HLA-DR and decrease of CD38, indicating continuous maturation of HL-60 cells to granulocytes and redifferentiation trend to monocytes. The aggregation of cells declined and the cell fragments increased. Myeloperoxidase （MPO） and α-naphthol acetate esterase （ot-NAE） activity increased after NS-shRNA-2 transfection. Furthermore, some HL-60 cells changed from round to fusiform shape even to pseudopod. The cells of small volume and karyorrhexis increased. Conclusion Through blocking the expression of NS gene, NS-shRNA can inhibit the cell proliferation and induce the differentiation of HL-60 cells, weaken malignant degree of HL-60 cells and trigger cell apoptosis. NS-shRNA is of clinical potential in gene therapy for acute leukemia.