氟伐他汀对HL-60细胞凋亡的诱导作用及其机制

Induction of fluvastatin on apoptosis of HL-60 cells and its mechanism

目的:研究氟伐他汀(Flu)对人早幼粒细胞白血病HL-60细胞(HL-60细胞)凋亡的诱导作用及可能的机制,为其临床应用提供理论依据。方法:体外培养的HL-60细胞分为空白对照组、阳性对照组[全反式维甲酸(ATRA),10μmol·L-1]、Flu组(20μmol·L-1)和Flu(20μmol·L-1)加[甲羟戊酸(Mev),100μmol·L-1]组。HL-60细胞被处理后,应用ACETM分析系统检测caspase-3活性以确定细胞凋亡,Annexin V-FITC/PI双染后流式细胞仪分析细胞凋亡,DNA凝胶电泳评价DNA碎片阶梯状条带及透射电镜观察细胞的超微改变。结果:处理24h后,Flu组HL-60细胞caspase-3活性(A405,30.68±1.57)显著高于对照组(12.05±2.15,P<0.01),Flu加Mev组HL-60细胞caspase-3活性明显降低(14.62±1.36),接近对照组细胞的水平(P>0.05)。Annexin V-FITC/PI双染后流式细胞仪检测,与对照组比较,Flu组HL-60细胞凋亡率明显增加(4.24%vs10.76%,P<0.01),Flu加Mev组HL-60细胞凋亡率降低到5.11%,与对照组细胞凋亡率(4.24%)比较差异无显著性(P>0.05)。处理72h后Flu组HL-60细胞在琼脂糖凝胶电泳图上可观察到DNA碎片梯状条带。透射电镜观察显示:Flu组HL-60细胞胞体缩小,胞质浓缩,核染色质凝聚,呈块状聚集于核膜内侧,胞膜及细胞器完好。结论:Flu能诱导HL-60细胞凋亡,这种作用是Mev途径依赖的,提示抑制Mev途径可能是Flu诱导HL-60细胞凋亡的分子机制之一。

Objective To investigate the induction of fluvastatin （Flu）on apoptosis of human promyelocytic leukemia cells（HL-60cells）and its mechanism,and provide theoretical basis for clinical application of Flu. Methods The cultured HL-60cells in vitro were divided into blank control,positive control group（ATRA, 10μmol·L-1）,Flu（20μmol·L-1）group and Flu（20μmol·L-1）＋ mevalonate（Mev,100μmol·L-1） group.After HL-60cells were treated,the caspase-3activity was determined using CaspACETM Assay System for exploring the apoptosis,the apoptosis was analyzed by flow cytometry after annexin V-FITC/PI double staining, the laddered pattern of DNA fragments was evaluated by DNA agarose gel electrophoresis and the ultrastructural changes of HL-60cells were observed by transmission electron microscope.Results After Flu treatment for 24h, the caspase-3activity in HL-60cells was significantly higher（A405,30.68±1.57）than that in control cells（12.05±2.15,P0.01）.In HL-60cells co-treated with Flu and Mev,the caspase-3activity was markedly reduced （14.62±1.36）,nearing to the level in control cells（P0.05）.Flow cytometry showed that compared with control,the apoptotic rate in Flu-treated HL-60cells was significantly increased（4.24%vs 10.76%,P0.01）, and the apoptotic rate was decreased to 5.11%after HL-60cells were co-treated with Flu and Mev（P0.05）. DNA agarose gel electrophoresis revealed a laddered pattern of DNA fragments in Flu-treated HL-60cells after 72h treatment.In Flu-treated HL-60cells,electron microscopic study exhibited the diminished cell body,condensed cytoplasm and nuclear chromatin condensation aggregating under the nuclear membrane,and the intact cell membrane and organelle.Conclusion Flu can induce the apoptosis of HL-60cells and this effect is dependent on mevalonate（Mev）pathway,suggesting that the inhibition of Mev pathway may be one of molecular mechanisms for Flu-induced apoptosis of HL-60cells.