线粒体损伤在创伤弧菌诱导树突状细胞凋亡中的作用

Role of damaged mitochondria in Vibrio vulnificus induced dendritic cell apoptosis

目的:探讨线粒体损伤在创伤弧菌(Vibrio vulnificus,Vv)诱导树突状细胞(dendritic cell,DC)凋亡过程中的作用及其可能机制。方法:建立Vv 1.1758与DC2.4细胞混合培养模型。采用扫描电镜和透射电镜观察创伤弧菌侵入细胞方式和细胞线粒体病变情况。荧光探针DCFH-DA和Fluo-8/AM分别检测侵入细胞内活性氧(ROS)和Ca2+离子水平。流式细胞术检测细胞线粒体膜电位和细胞凋亡情况。采用Western blotting检测NF-κB p65和TNF-α蛋白的表达。结果:Vv 1.1758可诱导DC2.4细胞凋亡。Vv 1.1758以菌体一端与细胞表面结合的方式侵入细胞,侵入细胞的线粒体有明显病变,细胞内ROS和Ca2+水平升高,线粒体膜电位降低。共培育1 h,NF-κB p65蛋白即开始升高,5 h达高峰,6 h稍有下降;TNF-α蛋白则在共培育2 h开始增高,6 h达高峰。结论:线粒体损伤在Vv诱导DC凋亡中发挥作用,其作用机制可能与细胞内ROS和Ca2+水平升高、线粒体膜电位降低有关,NF-κB p65和TNF-α可能是细胞凋亡过程中的重要信号分子。

AIM： To investigate the role of damaged mitochondria in dendritic cell（ DC） apoptosis induced by Vibrio vulnificus（ Vv） and its possible mechanism. METHODS： DC2. 4 cells were co-cultured with Vv 1. 1758 strain.Fluorescent probes DCFH-DA and Fluo-8-AM were used to detect reactive oxygen species（ ROS） and intracellular Ca^2 ＋concentration in the invaded cells,respectively. The cellular apoptotic rates and mitochondrial membrane potential（ Δψm）were measured by flow cytometry. The expression of nuclear factor-kappa B p65（ NF-κB p65） and tumor necrosis factor-alpha（ TNF-α） was detected by Western blotting. RESULTS： Vv 1. 1758 induced DC2. 4 cell apoptosis. Vv 1. 1758 bacteria invaded into the DC2. 4 cells by binding with cellular membrane though the end of the body. In the invaded DC2. 4cells,the visible mitochondrial damage,elevated ROS and intracellular Ca^2 ＋levels,and declined Δψmwere presented. After 1 h of co-culture,NF-κB p65 began to rise and reached the peak at 5 h,and then slightly decreased at 6 h. The TNF-α level increased after 2 h of co-culture and reached the peak at 6 h. CONCLUSION： The damaged mitochondria play an important role in DC apoptosis induced by Vv,and its possible mechanism may associate with the elevation of ROS and intracellular Ca^2 ＋level,and the declined Δψm. Meanwhile,NF-κB p65 and TNF-α are potential critical signaling molecules in the process of apoptosis.