丙戊酸钠诱导髓系白血病细胞分化、凋亡作用及其机制研究

Vaproate induces differentiation and apoptosis of myeloid leukemia cells and the related mechanisms

目的研究组蛋白去乙酰化酶抑制剂丙戊酸钠(VPA)诱导髓系白血病细胞体外分化和凋亡作用及其机制。方法采用四甲基偶氮唑盐(MTT)微量酶反应比色法增殖抑制实验和锥虫蓝拒染法检测VPA对细胞增殖的抑制,流式细胞仪检测细胞表面分化抗原的表达和细胞凋亡,Western印迹法检测组蛋白H3的9位赖氨酸残基(H3-Ac-lys9)的乙酰化水平变化。结果VPA抑制U937和K562髓系白血病细胞增殖,并呈时间-剂量依赖效应。在VPA较低浓度下(0.5～1mmol/L)作用6d后,VPA诱导U937细胞分化,其细胞表面髓系分化抗原CD11b表达明显上调,细胞形态呈终末分化改变。VPA与全反式维甲酸和(或)粒细胞集落刺激因子联合应用,诱导分化作用更为显著。在VPA较高浓度下(2mmol/L),VPA诱导U937细胞磷脂结合蛋白结合力明显上升,细胞呈现凋亡状态。而同样条件下VPA不能诱导K562细胞出现显著的分化和凋亡。U937和K562细胞在H3-Ac-lys9的乙酰化水平存在差异。VPA能够上调U937细胞H3-Ac-Lys9乙酰化,而K562细胞未观察到类似调变作用。结论VPA诱导U937细胞分化和凋亡,其作用与U937细胞的H3-Ac-lys9乙酰化水平及其对VPA处理后的上调有关。

Objective To study the efficacy of sodium valproate （VPA）, a histone deacetylase inhibitor （HDACi）, in inducing differentiation and apoptosis of myeloid leukemia cells and its possible mechanisms. Method Cell viability and proliferation assays were performed using trypan blue staining and MTT methods. Differentiation and apoptosis markers were analyzed by flow cytometry. The protein level of acetylated histone H3 at lysine 9（H3-Ac-lysg） was detected by Western blot. Results VPA inhibited the proliferation and viability of U937 and K562 cells in a time-and dose-dependent manner. Low concentration（0.5-1 mmol/L） of VPA induced myeloid differentiation in U937 cells, which was demonstrated by up-regulation of CDllb and morphology. When combined with ATRA and/or granulocyte colony-stimulating factor（G-CSF）, the induction effect of VPA was remarkably enhanced. High concentration（2 mmol/L） of VPA induced apoptosis of U937 cells as detected by flow cytometry. No differentiation or apoptosis was observed in K562 cells under same condition. U937 and K562 cells presented a significant difference in baseline level of acetylated H3-Ac-lys9. VPA significantly up-regulated the acetylation of H3-Ac-lys9 in U937 cells, but not in k562 cells. Conclusion VPA can induce differentiation and apoptosis in U937 cells, which might be associated with the baseline level of acetylated H3-Ac-lys9 in U937 cells and the sensitivity of H3-Ac-lys9 acetylation after VPA treatment. （Shanghai Med J, 2007, 30：847-851）