马钱子碱对白血病细胞株HL-60的作用研究

Effects of brucine on chronic myeloid leukemia cell line HL-60

目的 研究马钱子碱对人类白血病细胞株HL-60的增殖抑制作用及诱导凋亡作用.方法 分别用0、40、80、160、320、640 μg/ml的马钱子碱培养HL-60细胞,24、48、72 h后用四甲基偶氮唑蓝（MTT）法检测各组细胞的生长抑制率并计算IC50值；用AO/EB染色法荧光显微镜下观察细胞的形态学变化；用Annexin V-FITC/PI双染流式细胞术检测细胞的凋亡及凋亡率；用PI单染流式细胞术检测细胞周期分布.结果 马钱子碱可明显抑制HL-60细胞增殖,其作用呈剂量和时间依赖性,马钱子碱作用24、48、72 h后的IC50值分别为211.82、107、83μg/ml.320μg/ml马钱子碱作用48 h后观察到典型细胞凋亡形态,大部分细胞核染色质被EB染成桔红色并呈固缩状或圆珠状凋亡形态.流式细胞术检测显示各浓度组的细胞凋亡率分别为（2.1±1.1）％、（21.3±1.2）％、（38.6±1.3）％、（58.5±4.1）％、（75.3±0.87）％、（66.2±0.75）％,在0～ 320 μg/ml范围内随马钱子碱作用浓度的增加凋亡率逐渐升高,640 μg/ml浓度组细胞已无早期凋亡与活细胞.细胞周期检测显示明显的凋亡峰,与对照组相比G0/G1期细胞明显减少,G2/M期细胞明显增多.结论 马钱子碱可明显抑制人类白血病细胞株HL-60的增殖,其机制可能是诱导细胞凋亡和阻滞细胞于G2/M期.

Objective To investigate the proliferation inhibition and the apoptosis induction effect of brucine on human chronic myeloid leukemia cell line HL-60 cells.Methods HL-60 cells were exposed to various dosages of brucine 24,48,72 h respectively,MTT method was used to assay the growth inhibition effect of brucine on HL-60 cells and the IC50 of brucine was evaluated at the same time.The morphology was observed by AO/EB stains.The cell apoptosis and cell cycle was tested by flow cytometry with Annexin V-FITC/PI double staining and PI labeling respectively.Results The results indicated that the brucine displayed significant anti-proliferative effect on HL-60 cells in a dose-and time-dependent manner,with IC50 value of 211.8 μg/ml（24 h）,107 μg/ml（48 h）,83 μg/ml（72 h）respectively.The most significant inhibition was observed at 320 μg/ml for 48 h.In this condition,apoptosis morphology was induced by brucine with nuclear chromatin condensation,most of the nuclears were orange stained and condensation-like or bead-like,which was consistent with the Annexin V-FITC/PI results.The cell apoptosis rates were（2.1±1.1）％,（21.3±1.2）％,（38.6±1.3）％,（58.5±4.1）％,（75.3±0.87）％and（66.2±0.75）％in different dose of brucine,respectively.At the same time,the cell cycle analysis results showed that the cell ratio in G0/G1 phase was decreased while in G2/M and sub-G0 phases was increased,comparing with blank control group.Conclusion Brucine can inhibit cell growth dramatically,which may be related to the cell apoptosis and the cell cycle arrest.