蟾毒它灵通过线粒体途径诱导急性髓系白血病 HL-60细胞凋亡的实验研究

目的研究蟾毒它灵对急性髓系白血病HL-60细胞的作用及其机制,为急性髓细胞白血病的治疗提供新的思路。方法用终浓度为0、40、80、160μg/mL的蟾毒它灵作用于对数生长期的HL-60细胞24 h后,用CCK-8试剂盒检测细胞增殖情况,通过透射电子显微镜观察细胞内线粒体形态,蛋白免疫印迹法检测Bax、Bcl-2、Cleaved-Caspase-9基因表达情况,并用正常的外周血B淋巴细胞作为对照组观察该化合物对正常细胞的毒性作用。实验结果用SPSS 22.0统计学软件录入数据并进行检验,P<0.05为差异有统计学意义。结果蟾毒它灵能够抑制HL-60细胞增殖并诱导其凋亡,且对正常细胞毒性作用相较于癌细胞小,差异有统计学意义(P<0.05);随着药物浓度增加,细胞核异形,出现核膜分离,异染色质明显增多,线粒体伴有水肿扩张,部分线粒体脊消失模糊、空泡化,而对照组线粒体形态较好;随着药物浓度增加,Bax/Bcl-2比例增加,Cleaved-Caspase-9基因表达逐渐增加,差异有统计学意义(均P<0.01)。结论本实验结果表明蟾毒它灵对急性髓系白血病HL-60细胞这种非实体肿瘤细胞也有抑制作用,并且对正常细胞影响较小,其作用机制主要是通过线粒体途径诱导肿瘤细胞凋亡。

Induction of apoptosis in acute myeloid leukemia HL-60 cells by bufotalin via mitochondrial pathway

Objective To study the effect of bufotalin on acute myeloid leukemia HL-60 cells and its mechanism, so as to provide new ideas for the treatment of acute myeloid leukemia. Methods The HL-60 cells in the logarithmic growth phase were treated with quercetin at a final concentration of 0, 40, 80, and 160 μg/mL for 24 h, and the cell proliferation was detected by CCK-8 kit. The mitochondrial morphology was observed by transmission electron microscopy. The expressions of Bax, Bcl-2 and Cleaved-Caspase-9 genes were detected by Western blotting. The toxic effects of the compounds on normal cells were observed by using normal peripheral blood B lymphocytes as control group. The results of the experiment were recorded with SPSS 22.0 and tested. P<0.05 was considered statistically significant. Results Bufotalin could inhibit the proliferation of HL-60 cells and induce its apoptosis, and its normal cytotoxicity is smaller than that of cancer cells, with statistical significance (P<0.05). With the increase of drug concentration, nuclear showed abnormalities, nuclear membrane showed separation, and heterochromatin increased significantly, along with mitochondria with edema expansion, fuzzy disappearance of some mitochondrial ridges and vacuolization, while the control mitochondria morphology was better. When the proportion of Bax/Bcl-2 increased, Cleaved-Caspase-9 gene expression increased gradually, and the difference was statistically significant (all P<0.01). Conclusion The results of this experiment indicate that bufotalin also inhibits non-solid tumor cells of acute myeloid leukemia HL-60 cells, and has less impact on normal cells. The mechanism of its action is mainly to induce tumor cell apoptosis through mitochondrial pathway.