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目的:观察大萼香茶菜甲素(Macrocalyxin A,MA)体外对急性早幼粒细胞性白血病(NB4细胞)增殖抑制、诱导凋亡及诱导分化的作用,并探讨其作用机制。方法:将不同浓度MA在体外与NB4细胞共同培养,采用细胞计数、MTT细胞活性检测体外观察MA对NB4细胞增殖抑制作用;采用细胞形态观察、Hoechst33258荧光染色、DNA亚二倍体检测和Annexin V/PI双染色方法研究MA诱导NB4细胞凋亡作用;采用细胞形态学观察、NBT试验、细胞免疫表型CD11b检测等方法研究MA诱导NB4细胞分化作用;采用流式细胞技术分析凋亡调控基因Bcl-2、bax、bad、Fas、p53表达情况和线粒体膜蛋白、线粒体跨膜电位(△ψm)的改变探讨MA体外诱导NB4细胞凋亡和分化机制。实验设置空白对照和全反式维甲酸(ATRA)阳性对照。结果:不同浓度MA在体外对NB4细胞增殖有明显抑制作用,呈现剂量和时间的量效关系;较低浓度MA作用48h后可以诱导NB4细胞分化,较高浓度MA则显著地促进细胞凋亡;在凋亡调控基因中,Bcl-2、Fas表达无明显改变,bax、bad、Bcl-2/bax、p53随着药物作用浓度的增高表达逐渐增高;线粒体膜蛋白Apo2.7随着药物作用浓度和作用时间的增加表达逐渐增高,线粒体膜电位则逐渐下降。结论:MA在体外能明显抑制NB4细胞增殖,诱导细胞分化;一定浓度药物能够通过bax、bad等凋亡调控基因作用于线粒体诱导细胞凋亡。
OBJECTIVE: To observe the effects of Macrocyxin A (MA) on the proliferation, apoptosis induction and differentiation of acute promyelocytic leukemia (NB4) cells in vitro and to explore its mechanism. Methods: Different concentrations of MA were co-cultured with NB4 cells in vitro. The cell proliferation and proliferation of NB4 cells were observed by MTT assay and MTT cytotoxicity assay. Hoechst33258 fluorescence staining, DNA sub-diploid assay and Annexin V / PI double staining method was used to study the apoptosis of NB4 cells induced by MA. The differentiation of NB4 cells induced by MA was studied by morphological observation, NBT assay and CD11b immunocytochemistry. Flow cytometry was used to analyze the expression of apoptosis-regulating gene Bcl 2, bax, bad, Fas, p53 and the changes of mitochondrial membrane protein and mitochondrial transmembrane potential (△ ψm) were detected to investigate the mechanism of apoptosis and differentiation induced by MA in NB4 cells. The experiment set up blank control and all-trans retinoic acid (ATRA) positive control. Results: Different concentrations of MA significantly inhibited the proliferation of NB4 cells in vitro, showing the dose-time-dose-effect relationship. The differentiation of NB4 cells induced by MA at a lower concentration for 48h and the apoptosis induced by MA increased significantly. The expression of Bax, bad, Bax, bad, Bcl-2 / bax and p53 increased gradually with the increase of the drug concentration in the apoptosis regulatory genes. The mitochondrial membrane protein Apo2.7 with the concentration of the drug And the increasing expression of action time gradually increased, the mitochondrial membrane potential was gradually decreased. Conclusion: MA can obviously inhibit the proliferation of NB4 cells in vitro and induce cell differentiation. A certain concentration of drugs can induce mitochondria apoptosis through the regulation of bax, bad and other apoptosis regulatory genes.