大萼香茶菜甲素对急性早幼粒细胞性白血病细胞体外作用的影响

Effect of macrocalin A on NB4 Leukemia Cells in Vitro

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 investigate the effect of macrocalin A （MA） on NB4 leukemia cell in vitro. Methods： In vitro, NB4 ceils were incubated with MA, the cells＇growth was tested by cell counting, MTT assay. Cell morphology, Hoechst 33258 fluorescence staining, DNA content, cell cycle analyzation and Annexin-V/PI were used to investigate the apoptosis of NB4. The differentiation of NB4 was analyzed by cell morphology, cytometry and nitrobhie tetrazdium （NBT） test. The expressions of Bcl-2, bax, bad, Fas, p53 and mitochondrial membrane protein （APO2.7） were analyzed by flow cytometry. The mitochondrial transmembrancepotential （△ψm） was labeled by dihydrorhodamin 123. There were negative control and ATRA control in this test. Results： Different concentrations of MA could obviously inhibit the growth of NB4 cells with time and dose dependence. The cells were differentiated by low concentration of MA 48 hours later, while apoptosis were significantly induced by higher concentration of MA. The expressions of Bcl-2, Fas were no significant differences, while the expressions of bax, bad, Bcl-2/bax, p53 and Apo2.7 increased and △ψm decreased with time and dose dependent manner. Conclusion： In vitro, NB4 cells＇proliferation inhibition and differentiation could obviously induced by MA. The mechanism probably associated with its up-regulation of bax, bad, having the effect of opening the mitochondrial permeabihtytransition pore, reducing △ψm, finally inducing apoptosis,