蟾蜍灵对HL-60细胞的生长抑制及凋亡诱导作用

The mechanisms of inhibitory effect of bufalin on human leukemia cells

目的 研究中药蟾酥有效成分蟾蜍灵 (bufalin)对人白血病细胞的作用及其机制。方法 应用MTT比色法观察蟾蜍灵对细胞的抑制作用 ,荧光显微镜和透射电镜观察细胞结构的改变 ,DNA凝胶电泳、原位缺口标记法和流式细胞术等分析细胞凋亡。结果 ①蟾蜍灵明显抑制HL 6 0细胞的生长 ,半数抑制浓度 (IC5 0 )约为 0 .0 2 5 μmol L ;②典型的细胞形态学改变、DNA片段化和亚G1 峰的检出等证实蟾蜍灵能诱导白血病细胞凋亡 ;③ 0 .0 10 μmol L蟾蜍灵作用 2 4h即可明显诱导白血病细胞凋亡 ,凋亡率呈剂量和时间依赖性 ;④经蟾蜍灵作用后 ,细胞阻滞于S期 ,继而凋亡 ;⑤蟾蜍灵诱导的细胞凋亡能被核酸内切酶抑制剂氯化锌抑制 ,而不受蛋白质合成抑制剂放线菌酮的影响。结论 蟾蜍灵对白血病细胞有极强的诱导凋亡作用 ,主要作用于S期细胞 ,该作用不受蛋白质合成抑制剂的影响。

Objective To study the mechanism of bufalin on human leukemia cell inhibition. Methods HL 60 cells were treated with bufalin at different concentrations. The growth inhibition was analysed by MTT assay, cell apoptosis by light microscopy, transmission electron microscopy, flow cytometry, TUNEL labeling method and agarose gel electrophoresis. Results ①Treatment of HL 60 cells with bufalin remarkably inhibited the cell growth, the IC 50 value of bufalin for HL 60 cells was 0.025?μmol/L. ②Apoptosis of HL 60 cells could be efficiently induced by bufalin at concentration of 0.010?μmol/L or higher. ③Bufalin induced apoptosis of HL 60 cells in a dose and time dependent manner. ④With G 1 phase cells decreasing, S phase cells increased, and then apoptotic cells increased with a diminution of S phase cells. ⑤Bufalin induced apoptosis of HL 60 cells was inhibited by ZnCl 2, an inhibitor of endonuclease, but not by cycloheximide, an inhibitor of protein synthesis. Conclusion Bufalin could efficiently induce apoptosis of HL 60 cells, especially the S phase cells.