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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 effect and mechanism of bufalin, an effective component of Chinese wolfberry, on human leukemia cells. Methods MTT colorimetric method was used to observe the inhibitory effect of bufalin on the cells. Fluorescence microscopy and transmission electron microscopy were used to observe the changes of cell structure. Cell apoptosis was analyzed by DNA gel electrophoresis, in situ gapping and flow cytometry. Results 1 The growth of HL 60 cells was significantly inhibited by bufalin, the half inhibitory concentration (IC50) was about 0.25 μmol L; 2 typical cell morphology changes, DNA fragmentation, and detection of sub-G1 peaks were confirmed. Fuling can induce apoptosis of leukemia cells; 3 0 .10 10 μmol L Bufalin can significantly induce apoptosis of leukemia cells for 24 h, and the apoptosis rate is dose- and time-dependent; 4 After blockade, cell block occurs. In S phase, followed by apoptosis; apoptosis induced by 5-azulene can be inhibited by the endonuclease inhibitor zinc chloride, but not by the protein synthesis inhibitor cycloheximide. Conclusion The bufalin has a strong apoptosis-inducing effect on leukemia cells, mainly acting on S-phase cells, and this effect is not affected by the inhibitor of protein synthesis.