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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 effects of NSC-specific short hairpin RNA (shRNA) on differentiation antigen and biological properties of HL-60 leukemia cells, and to investigate the biological functions of NS Acute leukemia and the possibility of gene therapy. METHODS: The transfected HL-60 cells were transfected with the NS-shRNA short hairpin RNA (NS-shRNA) by specific transfection reagent and the gene blocking effect was determined by RT-PCR. The cell proliferation and flow cytometry Cell differentiation antigen was detected by FCM. Cell shape and growth state were observed by morphology. Cell size and particle size were determined by hematology analyzer. Results Both NS-shRNA and NS-shRNA could significantly inhibit NS gene expression, with the inhibition rates of 37.82% and 71.88%, respectively. NS-shRNA significantly inhibited the proliferation of HL-60 cells in a time- and concentration-dependent manner. And 10 nmol / L the best concentration. After transfection, the differentiation antigens such as CD11b, CD33, CD14, CD64 and HLA-DR were increased and CD38 was decreased, showing that the granulocytic lines continued to mature and re-differentiated into mononuclear lines. The cell agglomeration decreased, the number of fragments increased and some cell morphology Into a fusiform and long tail, the nucleus was dense, the depression, myeloperoxidase (MPO) and α-NAE activity increased; the size and size analysis of small cells and nuclear fragmentation Increased cells. Conclusion NS-shRNA can inhibit the proliferation and differentiation of HL-60 cells by blocking the expression of NS gene, change the biological properties of cells, reduce the degree of cell malignancy and induce apoptosis. In NSCLC, Gene has potential clinical value.