凋亡细胞细胞膜和线粒体的动态变化

Kinetics of plasma membrane and mitochondrial alterations in HL-60 cells undergoing apoptosis

应用拓扑异构酶抑制剂VP-16及化学毒物叠氮钠分别诱导HL-60细胞凋亡及坏死,在0、2、4、8、16及24h各时间点,应用Hoechst33258染色,荧光显微镜及透射电镜对核形态进行观察,通过流式细胞术检测二乙酸荧光素(FDA)、罗丹明123(Rh123)和碘化丙啶(PI)荧光强度的变化,观察细胞凋亡及坏死过程中细胞膜通透性和线粒体膜电位的动态变化。结果显示:HL-60细胞在VP-16处理4h时核形态开始变化,有核浓缩的细胞比例在8h达高峰,然后下降;核碎裂细胞的比例在24h达高峰,约占80%,表明核浓缩发生在核碎裂之前。线粒体跨膜电位在8h后渐渐降低,16h可见明显降低。坏死细胞的线粒体膜电位在4h降低50%,细胞膜通透性也在8h之后出现变化。随处理的时间延长,FDA荧光强度进一步降低,对PI的摄取渐渐升高。结果提示,细胞凋亡过程中细胞膜通透性逐渐增高,线粒体膜电位降低。这两个指标能反映细胞凋亡及其程度,结合对细胞核形态观察,能更准确、可靠地反映细胞凋亡的发展变化。

In the present study, VP16,an inhibitor of topoisomerase Ⅱ, and NaN 3, a chemical toxic substance, were used to induce apoptosis and necrosis of HL 60 cells, respectively. Cells were examined by transmission electron microscopy and by fluorecence micrscopy after staining with Hoechst 33258 at 0,2,4,8,16 and 24h of culture. Alterations of fluorescent intensity produced by FDA, Rh 123 and PI were measured by flow cytometry, which reflects sequential changes on plasma membrane permeability and the potential of mitochondrial membrane. The results indicated that the nuclear morphologic alteration of cells treated with VP 16 began at 4 hour of culture and the cells with condensed nucleus culminated at 8h of culture and then reduced with no drop in cell counts, while the percentage of cells with fragmented nuclei reached its maximum at 24h of culture accounting for some 80%. The potential of mitochondrial membrane of of apopototic cells decreased gradually from 8h of culture and showed obvious decline at 16h of culture.In necrotic cells, the potential decreased by 50% at 4h of culture which indicates an earlier and more rapid decline than that in apopototic cells. Alteration of plasma membrane permeability appeared at 8h of culture and showed a steady increase over time. It was concluded that plasma membrane permeability and mitochondrial membrane potential could reflect the development and degree of apoptosis, and the combination of these two criteria with nuclear morphology revealed by staining with Hoechst 33258 would be a simple and reliable assay for apoptosis and its progression.