创伤弧菌诱导树突状细胞凋亡的过程观察

Observation for the process of Vibrio vulnificus inducing dendritic cell apoptosis

目的实验观察创伤弧茵（Vibrio vulnifwus，Vv）诱导鼠树突状细胞（dendriticcell，DC）凋亡的过程。方法建立小鼠树突状细胞（DC2．4株）与创伤弧菌（Vvl．1758株）混合培养模型，DAPI荧光染色分析细胞凋亡的形态特征，DNALadder检测凋亡细胞的DNA片段化水平分析，Annex—inVFITC／PI染色分析DC2．4细胞凋亡率，分光光度法测定caspase-3、caspase-8活性，JC-1荧光标记检测线粒体膜电位（A山m）变化。结果Vvl．1758株与DC2．4细胞混合培养4h时DAPI荧光染色出现典型的凋亡特征——染色质浓缩及边缘化；DNA琼脂糖凝胶电泳出现凋亡条带；2、4、6h细胞凋亡率分别为（37．8±9．8）％、（54．3±12．7）％和（68．2±14．6）％；1、2、4h线粒体膜电位（△ψm）分别下降了7．1％、16．1％与46．7％；caspase-8活性在1．5h增高，2h达高峰（2．48±0．19）U／μg，而caspase-3活性于3h开始增高，4h达高峰（1．91±0．16）U／μg。结论创伤弧菌诱导树突状细胞可通过线粒体膜电位下降及激活caspase-8启动子两条途径，最终活化效应因子caspase-3，促使细胞凋亡发生。

Objective To observe the process of Vibrio vulnificus inducing dendritic cell strain ap- optosis. Methods We established the mixed culture model of mouse dendritic cell （ DC 2.4 strain） and Vibrio vulnificus（ Vvl. 1758 strain）, analyzed morphological characteristics of cell apoptosis by DAPI fluores- cence staining, detect DNA fragmentation level of apoptosis cells by DNA Ladder assay, analyze DC2.4 ap- optosis rate by Annexin V FITC/PI staining, determine activities of caspase-3 and caspase-8 by means of spectrophotometric method and detect changes of mitochondrial transmembrane potential （ A ~m） by JC-1 fluorescence labeling. Results After Vvl. 1758 strain and DC2.4 cell were mixed and cultured for 4 h, DAPI fluorescence staining showed typical apoptosis characteristics--chromatin condensation and marginal- ization; DNA agarose gel electrophoresis showed apoptosis band; apoptosis rates at 2, 4 and 6 h were re- spectively （37.8 ＋9.8 ） %, （54.3 ＋ 12.7 ） % and （68.2_＋ 14.6 ） % ; Mitochondrial transmembrane potentials （△ψm） at 1 h, 2 h and 4 h reduced by 7.1% , 16.1% and 46.7% respectively; caspase-8 activity in- creased at 1.5 h and reached the peak at 2 h [ （2.48±0.19） U/μg], while caspase-3 activity started to in- crease at 3h and reached the peak at 4 h [ （1.91±0.16） U/μg ]. Conclusion Vibrio vulnificus could in- duce dendritic cells by two pathways： reducing mitochondrial transmembrane potential and activating caspase-8 promoter and finally activate effector easpase-3 to promote apoptosis.