三氧化二砷联合苦参碱抑制K562细胞增殖及机制研究

Effects of Arsenic Trioxide in Combination with Matrine in inhibiting the K562 Cells Proliferation and Its Mechanism

目的探讨三氧化二砷联合苦参碱对慢性髓系白血病细胞株K562增殖的影响及可能机制。方法用不同浓度的三氧化二砷单药(0.75、1.5、3、6、8μmol/L),苦参碱单药(0.5、0.75、1.0、1.25、1.5g/L)及三氧化二砷(3μmol/L)+苦参碱(0.75g/L)两药联合分别作用于K562细胞,CCK-8法测定细胞增殖抑制率;流式细胞术检测细胞周期,免疫印迹法(Westernblot法)检测cyclin D1、CDK4、p^(21)蛋白表达的变化。结果不同浓度苦参碱及三氧化二砷均可抑制K562细胞增殖,且随作用时间延长和浓度增加而明显(P<0.05),两药联合应用后抑制作用更显著(P<0.05);与对照组比较,苦参碱阻滞K562细胞于G_1期,三氧化二砷阻滞K562细胞于G_2期,苦参碱可增强三氧化二砷对K562细胞周期的阻滞作用;与单药组及对照组相比,两药联合后K562细胞p^(21)蛋白表达水平上调(P<0.05),cyclin D1、CDK4蛋白表达水平明显下降(P<0.05)。结论苦参碱可增强三氧化二砷对K562细胞的增殖抑制作用,阻滞细胞于G_2期,其机制可能与p^(21)蛋白表达增加及cyclin D1、CDK4蛋白表达减少相关。

Objective To explore the effects of arsenic trioxide（ATO） in combination with matrine（Mat） on the cell proliferation of chronic myeloid leukemia cell line K562 and its possible mechanisms. Methods Cells were treated with different concentration of ATO alone （0.75,1.5,3,6,8μmol/L）and different concentration of Mat alone（0.5,0.75,1.0,1.25,1.5g/L） and the combination of ATO（3μmol/L）and Mat（0.75g/L） separately. The cell proliferation inhibit rate of K562 cells were measured by CCK-8 method.The cell cycle distribution were examined by flow cytometry.The expression level of cyclinD1,CDK4 and p21 protein were detected by Westernblot. Results Both the ATO and Mat can inhibited the cell proliferation of K562 cells in a time-and dose-dependent manner（P〈0.05）.The combination of ATO and Mat has synergistic effects（P〈0.05）. Compared with control group,the cell cycle of K562 cells was blocked in G1 phase by Mat,and blocked in G2 phase by ATO. Matrine can enhance the cell cycle arrest effect of ATO in K562 cells. Compared with single drug-treatment group and control group,the combination treatment up-regulated the expression level of p21 protein（P〈0.05）,while the expression level of cyclinD1 and CDK4 protein were down-regulated （P〈0.05）. Conclusion Mat can enhance the effect of ATO on inhibiting the proliferation of K562 cells,and blocked the cell cycle in G2 phase.The action of blocking cell cycle is closely associated with its up-regulating the expression level of p21 protein,down-regulating the expression of the expression level of cyclinD1 and CDK4.