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核仁磷酸蛋白基因(nucleophosmin,NPM1)突变在急性髓系白血病的发生发展中发挥着重要作用,而与白血病分化阻滞的关系尚未完全阐明。为探讨NPM1基因突变对白血病细胞体外分化的影响,将携带NPM1 A型突变(NPM1-mA)的表达质粒载体pEGFPC1-NPM1-mA转染白血病K562细胞系,构建稳定表达NPM1-mA蛋白的细胞株(K562 mA),同时设立野生型NPM1转染组(K562 wt)、空载体转染组(K562 C1)和未处理组(K562)为对照。利用豆蔻酰佛波醇乙酯(PMA)诱导各组细胞分化,瑞氏–吉姆萨染色观察细胞分化的形态改变,计算诱导分化率;相差显微镜计数贴壁细胞数量;流式细胞术分析细胞表面分化抗原CD41的表达。结果显示,PMA作用72 h后,与对照组相比,K562 mA组细胞的诱导分化率及贴壁细胞数明显降低(P<0.05);同时,CD41的表达受到显著抑制(P<0.01)。提示NPM1基因突变能够阻滞白血病细胞系K562的体外分化。
The nucleophosmin (NPM1) mutation plays an important role in the development of acute myeloid leukemia, but its relationship with the differentiation and arrest of leukemia has not been fully elucidated. To investigate the effect of NPM1 gene mutation on the differentiation of leukemia cells, NPM1-NP expression vector pEGFPC1-NPM1-mA was transfected into leukemia K562 cell line to construct a cell line stably expressing NPM1-mA protein (K562 mA). At the same time, wild-type NPM1 transfection group (K562 wt), empty vector transfection group (K562 C1) and untreated group (K562) were established as controls. Differentiation of cells was induced by myristylphorbol ethyl ester (PMA). Wright-Giemsa staining was used to observe the morphological changes of the cells. The rate of induced differentiation was calculated. The number of adherent cells was counted by phase contrast microscopy. The cell surface was analyzed by flow cytometry Differentiation antigen CD41 expression. The results showed that compared with the control group, the differentiation and the number of adherent cells in K562 mA group were significantly decreased (P <0.05) at 72 h after PMA treatment. Meanwhile, the expression of CD41 was significantly inhibited (P <0.01). These results suggest that NPM1 gene mutation can block the differentiation of leukemia cell line K562 in vitro.