细胞周期特异性细胞凋亡的形态学观察

MORPHOLOGICAL OBSERVATION OF THE CYCLE SPECIFIC APOPTOSIS

目的观察喜树碱诱导Molt-4细胞凋亡形态的细胞周期时相性变化,建立一种新的细胞周期特异性细胞凋亡分析方法。方法以凋亡诱导剂喜树碱(终浓度为0.15μmol/L)影响下的Molt-4细胞为检测对象,应用流式细胞术分析细胞凋亡及细胞周期的变化;用琼脂糖凝胶电泳的方法探测断裂的DNA片段存在,进一步证实细胞凋亡的存在;碘化丙啶(PI)染色后,再应用流式分选术结合激光扫描共聚焦显微镜(laser scanning confocal microsope,LSCM)观察各期细胞形态从而对已凋亡细胞进行形态学确认。结果①琼脂糖凝胶电泳可见清晰的DNA梯形条带(DNAladder);②喜树碱导致Molt-4细胞周期的S期水平明显下降,并伴随有一明显的细胞凋亡峰;③只在S期细胞中可见到已凋亡细胞。以上说明喜树碱可以诱导Molt-4细胞凋亡,并细胞凋亡发生在细胞周期中的S期,与传统的结论一致。结论通过DNA直方图分选后细胞凋亡形态学分析可以对细胞凋亡的细胞周期特异性进行检测。这种方法使细胞周期特异性凋亡的分析更加可靠全面。

Objective To observe the morplaological changes of Molt-04 cells in tne specific phase of the cell cycle induced by camptothecin （CPT） , in order to establish a new method for the analysis of cell cycle specific apoptosis. Methods Molt-4 cells influenced by CPT （0. 15μmol/L ） capable of inducing apoptosis were used as subjects. Changes in the cell apoptosis and the cell cycle were analysed by using flow cytometry. DNA fragments of apoptosis were tested by using agarose gel eletrophoresis to prove the cell apoptosis. After staining with propidium iodide （PI）, the specific phase of the drug-induced cell apoptosis in the cell cycle was explored by flow cytometry sorting combined with confocal microscopic pictures of the cells in different phases of the cell cycle. Results ①The DNA ladder of agarose gel eletrophoresis could be seen clearly. ② There was a Sub-G1 in the drug-induced cell cycle and the level of S phase was declined in the cycle. ③The apoptotic cells were only observed in S phase. The results are consistent with the traditional conclusion that camptothecin can induce the apoptosis of Molt-4 cells, and in S phase only. Conclusion The cell cycle specificity of apoptotic cells can be detected by DNA histogram sorting combined with morphological analysis. This method makes the analysis of the cell cycle specific apoptosis more reliable and full-scale.