结核分枝杆菌诱导小鼠树突状细胞凋亡及caspase-3、-8活化对凋亡的影响

Effects of Mycobacterium tuberculosis on apoptosis of mouse dendritic cells and activation of caspase-3,caspase-8

目的:研究结核分枝杆菌(Mycobacterium tuberculosis,MTB)诱导小鼠树突状细胞株(DC2.4)凋亡及caspase-3、-8活化对凋亡的影响。方法:建立小鼠树突状细胞DC 2.4的人结核分枝杆菌H37Rv细胞混合培养模型。采用DAPI荧光染色法和透射电镜分析凋亡DC 2.4细胞的形态特征;通过DNA琼脂糖凝胶电泳进行DNA片段化分析,FITC-Annexin V/PI荧光标记流式细胞术检测DC 2.4细胞凋亡情况;分光光度法检测H37Rv诱导DC 2.4细胞凋亡过程中caspase-3、-8活性变化。结果:H37Rv与DC 2.4细胞混合培养2 h后,即有细菌侵入,培养4、6、8、10、12 h后,细菌侵入现象更明显,侵入率分别为(16.1±4.3)%、(35.8±5.1)%、(50.2±5.7)%、(58.3±6.2)%和(65.9±6.9)%。H37Rv与DC 2.4细胞混合培养6 h后,DAPI染色荧光显微镜、透射电镜观察均发现DC 2.4细胞发生凋亡并出现典型的凋亡特征———染色质浓缩及边缘现象;DNA琼脂糖凝胶电泳出现特征性的凋亡条带。H37R与DC 2.4细胞混合培养6 h、12 h和24 h后,细胞凋亡率分别为(6.4±2.5)%,(11.8±5.3)%和(31.1±8.7)%。caspase-3、-8活性增高,caspase-3活性于10 h达高峰(2.01±0.09)U/μg,而caspase-8活性则在6 h达高峰(2.40±0.07)U/μg,两者与对照组相比均具有统计学意义(P<0.05),且caspase-8激活先于caspase-3。结论:结核分枝杆菌可诱导树突状细胞发生凋亡,其诱导凋亡的机制可能与caspase-3、-8活化有关。

Objective: To investigate the effects of Mycobacterium tuberculosis on apoptosis of mouse dendritic cells(DC 2.4) and the activation of caspase-3,caspase-8. Methods: Mycobacterium tuberculosis H37Rv strain was co-cultured with DC 2.4 cells.The morphological changes of DC 2.4 cells were observed with fluorescence microscope after DAPI staining and transmission electron microscope.The apoptosis of DC 2.4 cells were examined by DNA agarose gel electrophoresis.The activities of caspase-3 and caspase-8 were detected by colorimetric assay. Results: Bacterial invasion was observed while DC 2.4 cells and H37Rv were co-cultured for 2 h;and the rates of invasion were(16.1±4.3)%,(35.8±5.1)%,(50.2±5.7)%,(58.3±6.2)% and(65.9±6.9)% at 4,6,8,10,12 h,respectively.The phenomenon of nuclear condensation and marginalization were shown by DAPI staining and transmission electron microscope in DC 2.4 cells at 6 h of co-cultivation with H37Rv.The characteristic bands of apoptosis by DNA electrophoresis were detected.The activities of caspase-3 and caspase-8 were increased in a time-dependent manner.The rates of DC 2.4 cell apoptosis were(6.4±2.5)%,(11.8±5.3)% and(31.1±8.7)% at 6 h,12 h and 24 h after co-cultivation with H37Rv,respectively.The maximal activities of intracellular caspase-3 and caspase-8 at 10 h and 6 h were(2.01±0.09) U/μg and(2.40±0.07)U/μg,respectively,which was significantly different compared with the control groups(P<0.05).The activation of caspase-8 was earlier than that of caspase-3. Conclusion: Mycobacterium tuberculosis can induce the apoptosis of DC 2.4 cells,which is associated with the activation of intracellular caspase-3 and caspase-8.