针对病毒性心肌炎的细胞凋亡研究

Cell apoptosis of viral myocarditis derived by Coxsackievirus

目的探讨柯萨奇病毒致细胞死亡时是否存在凋亡,以及凋亡百分率。方法柯萨奇病毒感染心肌细胞和HeLa细胞以后,应用TUNEL原位标记、DNALadder、Hoechst33258染色和Annexin-V/PI染色观察细胞凋亡发生情况;并通过流式细胞仪分析细胞凋亡发生率。结果Hoechst33258染色发现病毒感染后的细胞核出现强亮度的蓝色荧光,可观察到凋亡细胞的典型变化;Annexin-V/PI染色可见HeLa细胞膜显强绿色荧光,细胞核显强红色荧光;感染组细胞DNA出现阶梯状条带影。通过流式细胞仪检测被PI染色的细胞DNA,发现病毒感染组出现凋亡峰,凋亡率为49.5%。结论柯萨奇病毒致病毒性心肌炎时细胞不仅存在凋亡,而且是细胞主要死亡形式。

[Objective] To investigate the occurrence of cell apoptosis in cultured cells infected by Coxsackievirus. [Methods] Cardiac myocytes and HeLa cells were cultured and subsequently infected by Coxsackievirus, then detected the occurrences of cell apoptosis by the methods of Hoechst33258 staini.ng, Annexin- V/PI staining, TUNEL and DNA Ladder. HeLa cells were grouped and infected by series concentration of virus, and the ratio of apoptosis was determined by flow cytometry. [Results] HeLa cells affected by CVB were displayed strong blue fluorescence in nueleolus after stained with Hoechst 33258, and displayed green and red fluorescence in membrane and nucleolus, respectively, after stained with Annexin-V/PI. TUNEL assay demonstrated that cell nuclei were highly condensed after treatment with CVB, and distinct DNA ladder could be detected after agarese gel electrophoresis. By using cytometry, file occurrence of apoptosis was exactly determined; the ratio of apoptosis reached 48.80% when the concentration of CVB was 100TCID50. But no significant apex of apoptosis could be detected if the concentration of CVB was too high. {Conclusion] Death cells infected by Coxsaekievirus are mostly areused by apoptosis.