B型流感病毒细胞凋亡作用的体外研究

ANALYSIS OF APOPTOSIS INDUCED BY INFLUENZA VIRUS B IN MDCK CELLS IN VITRO

[目的]研究B型流感病毒导致的体外培养狗肾传代细胞凋亡作用,初步探讨流感病毒细胞毒性发生机制。[方法]采用Hoechst 33258荧光染色观察形态学变化,DNA片段的琼脂糖凝胶电泳检测细胞凋亡,流式细胞术ANNEXINV-FITC加碘化丙啶(PI)双染定量检测细胞凋亡率和坏死率。[结果]加入不同滴度的B型流感病毒培养6小时后开始出现细胞核荧光强度增强,染色质沿核周分布现象。DNA电泳呈典型的"梯形"条带。流式细胞术检测发现,不同滴度流感病毒作用后,细胞凋亡率明显高于空白对照组,差异有显著性(P﹤0.05)。流感病毒滴度越高,凋亡率也越高,但当流感病毒滴度为1︰128时,细胞坏死率增加。滴度为1︰64的流感病毒诱导的细胞凋亡,随着作用时间的延长,凋亡率明显增高,作用18h达高峰,而后细胞凋亡率下降。[结论]体外培养条件下B型流感病毒能诱导MDCK细胞凋亡,并存在量-效与时-效关系。

[Objective] To inVeStigate whether influersea virus B can induce apoptosis in MDCK cells in vitro, and to explore the relationship between the meghanism and cytotoxicity with influenza vires tentativelyi [Methods] Cell morphology was observed by Hoechst 33258 fluorescent stain method, and DNA fragments were analyzed by agarose gel electrophoresis.Anti Annexin V FITC plus PI staining for spoptotic and necrotic rate was examined by flow cytometer. [ Results] After adding different Concentrations of influenza virus B to the cells for 6 hours, the fluoresent intensity Of nucleus increased, and the chromatin was condensed and distributed around the necleus DNA ladder was detected in some samples. Cell apoptotic rate in different concentration of influenza virus groups was significantly higher than that in blank Control group （P 〈 0.05）. With the increase of the concentration of intluenza virus; the apoptotic rate also increased. The optimal concentration of influenza virus B was 1 ： 64, When it was equal to 1 ： 128,the necrotic rate increased significantly. [Conclusion] Influenza virus B can induce apoptosis of MDCK cells in vitro with dose-effect andd time-effect relationship.