三氧化二砷诱导急性早幼粒白血病细胞凋亡的机制

The mechanism of apoptosis of acute promyelocytic leukemia cells induced by arsenic trioxide

目的探讨线粒体在三氧化二砷(As2O3)诱导急性早幼粒细胞白血病NB4细胞凋亡中的作用并观察线粒体基因组在该过程中的表达变化。方法采用荧光显微镜、流式细胞仪、细胞生长抑制实验、线粒体膜电位检测、RT-PCR等方法,观察As2O3对NB4细胞的诱导凋亡、生长抑制作用及线粒体基因差异表达变化。结果荧光显微镜观察显示,NB4细胞经As2O3处理48h后呈现出明显的凋亡形态学改变。As2O3在一定的浓度范围内对NB4细胞具有显著的生长抑制效应。流式细胞仪分析发现,NB4细胞经0.5、1、2、3μmol/LAs2O3处理后,其细胞凋亡率分别为5.02%、6.40%、28.40%、33.34%。以0.5、1、2、4、8μmol/LAs2O3分别处理NB4细胞48h后,流式细胞仪检测NB4细胞线粒体膜电位分别下降12.8%、21.6%、66.9%、83.7%、83.8%。对As2O3诱导NB4细胞凋亡过程中的13个线粒体基因组基因进行RT-PCR分析发现,COX2基因表达下调,其余12个基因表达无明显改变。结论As2O3在体外对NB4细胞具有显著的诱导凋亡及生长抑制效应,其诱导凋亡作用与线粒体膜电位下降密切相关;线粒体相关基因COX2的表达变化参与了As2O3诱导的NB4细胞凋亡过程。

Objective To investigate the effects and the gene expression of mitochondria on arsenic trioxide-induced apoptosis of acute promyelocytic leukemia （APL） NB4 cells. Methods NB4 cells were treated with As2O3. Fluorescent microscopy and flow cytometry were used and mitochondria membrane potential detection and RT-PCR were performed to observe the NB4 cell apoptosis, growth inhibitory effect and mitochondrial transmembrane potentials. Mitochondria genome primers were designed, and the expression of mitochondria genome at gene and protein levels was studied. Results Induced by As2O3, the NB4 cells showed typical morphological changes of apoptosis with a significant growth inhibitory effect. The ratios of NB4 cells apoptosis were 5.02%, 6.40%, 28. 40% and 33.34%, respectively, when treated with As2O3 in concentrations of 0.5μmol/L, 1μmol/L, 2μmol/L and 3μmol/L. When treated with As2O3 in 0.5μmol/L, 1μmol/L, 2μmol/L, 4μmol/L and 8μmol/L at 48h, the mitochondria potential of NB4 cells was decreased by 12.8%, 21.6%, 66. 9%, 83. 7% and 83.8%, respectively. After the NB4 cell apoptosis was induced by As2O3, RT-PCR assay was used to detect the expression of 13 genes of mitochondria genome. The expression of COX2 gene was down-regulated in this process, while no change was found in the expression of other 12 genes. Conclusions As2O3 has shown to exert a significant effect on promoting apoptosis and growth inhibition on APL cells. The apoptotic effect induced by As2O3 on NB4 cells is closely related to a decrease of mitochondrial membrane potential. The expression change in the mitochondria gene COX2 is involved in the As2O3-induced apoptosis of NB4 cells.