电镜和荧光显微技术在细胞凋亡研究中的应用

目的：探讨荧光显微技术在检测细胞凋亡中的作用。方法：应用拓扑异构酶Ⅱ抑制剂VP-16及化学毒物叠氮钠分别诱导HL-60细胞凋亡和死亡，用透射电镜和经Hoechst 33258染色的荧光显微镜对凋亡及死亡动态观察和定量分析。结果：HL-60细胞在VP16处理后，荧光显微镜下可见核浓缩、染色质凝集、核碎裂等凋亡特征；而叠氮钠诱导的细胞死亡则出现核溶解、核染色质弥散，但无上述改变；两者电镜的观察与荧光显微镜的改变一致；凋亡细胞及坏死细胞荧光显微镜下呈不同的形态特征。对处理的不同时间点细胞的荧光显微镜观察发现：处理后4h核形态开始变化，有核浓缩的细胞比例在处理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。结论：荧光显微镜技术可明确观察判断细胞凋亡及坏死，并可进行动态及定量分析，是良好的凋亡检测方法。

Continual and quantitative analysis of cellular apoptosis by electron and fluorescence microscope

OBJECTIVE: To evaluate the usage of fluorescent microscope in analyzing cellular apoptosis. METHODS: VP16 (the inhibitor of Top-II) and Azard Sodium(NaN3, the chemical toxic agent) were used to induce cellular apoptosis and necrosis respectively. The cellular changes were analyzed continually and quantitatively by transmission electromicroscope and fluorescent microscope stained by Hoechst 33258. RESULTS: Under the observation of fluorescent microscope and electromicroscope, HL60 cells showed pyknosis, chromatin aggregation and karyorrhexis (the features of apoptosis) in the induction of VP16; whereas the cells in the induction of NaN3 presented necrotic changes of karyolysis and chromatin diffusion. This indicated that there were different features in cellular apoptosis and necrosis under fluorescent microscope. In a time course observation of fluorescent microscope, the pyknosis began at 4 h after induction and peaked at 8 h, whereas karyorrheix reached 80% at 24 h, suggesting that the pyknosis preceded the karyorrheix. CONCLUSION: The fluorescent microscopic observation is a technique in continual and quantitative analysis of apoptosis, which may be used to distinguish the cellular apoptosis and necrosis.