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目的 :探讨蟾毒灵(bufalin)对伯基特(Burkitt)淋巴瘤细胞的影响。方法:体外培养人伯基特淋巴瘤细胞株Daudi,以0、5、10、20、40、80 nmol/L浓度蟾毒灵处理细胞,细胞计数试剂盒8(CCK-8)检测细胞抑制率,膜联蛋白(annexin)Ⅴ/碘化丙啶(PI)双染后,流式细胞仪检测细胞凋亡,蛋白质印迹(Western blotting)检测凋亡相关蛋白胱天蛋白酶及B细胞淋巴瘤(Bcl)-2、c-Myc的变化,实时定量(real-time)PCR检测c-Myc RNA水平的变化。结果:CCK-8检测结果显示,蟾毒灵对Daudi细胞增殖有明显的抑制作用,呈药物浓度依赖性,且经48 h处理后的抑制率明显高于24 h,半数抑制浓度(IC50)为(20.03±2.56)nmol/L;流式细胞检测结果表明,蟾毒灵可诱导Daudi细胞发生凋亡;蛋白质印迹检测结果显示,不同浓度蟾毒灵处理Daudi细胞48 h后,胱天蛋白酶3、9及多聚腺苷二磷酸核糖聚合酶1(PARP1)均出现明显剪切,并伴有Bcl-2及c-Myc癌基因的下调;实时定量PCR检测显示,c-Myc癌基因在m RNA水平也出现明显下调。结论:蟾毒灵能够诱导Burkitt淋巴瘤Daudi细胞株发生凋亡,下调的c-Myc及Bcl-2癌基因可能参与了这一过程。
Objective: To investigate the effect of bufalin on Burkitt lymphoma cells. METHODS: Human Burkitt’s lymphoma cell line Daudi was cultured in vitro. Cells were treated with bufalin at concentrations of 0, 5, 10, 20, 40 and 80 nmol / L, and cell counting kit 8 (CCK-8) , Annexin Ⅴ / propidium iodide (PI) double staining. Flow cytometry was used to detect apoptosis. Western blotting was used to detect apoptosis related protein caspase and B cell lymphoma ) -2, c-Myc changes in real-time PCR detection of c-Myc RNA levels. Results: The results of CCK-8 showed that Bufalin significantly inhibited the proliferation of Daudi cells in a drug concentration-dependent manner, and the inhibition rate after 48 h treatment was significantly higher than that of 24 h. The IC50 values were (20.03 ± 2.56) nmol / L. The results of flow cytometry showed that bufalin could induce apoptosis in Daudi cells. Western blotting showed that after treatment with different concentrations of bufalin for 48 h, the expression of caspase 3, 9 and poly (ADP-ribose) polymerase 1 (PARP1) were significantly cleaved, accompanied by the down-regulation of Bcl-2 and c-Myc oncogenes; real-time quantitative PCR showed that c-Myc oncogene in m RNA The level also significantly reduced. Conclusion: Bufalin can induce the apoptosis of Burkitt lymphoma cell line Daudi. Down-regulated c-Myc and Bcl-2 oncogenes may be involved in this process.