重建线粒体动态平衡对NK细胞NK2型活化影响

目的立于“线粒体功能紊乱-细胞异常活化”视角,探讨NK细胞NK2型活化机制。方法将NK-92MI细胞分为空白组、TSLP组、1、5、10μmol·L^-1 Mdivi-1组。ELISA法测定各组IL-4、IL-5、IFN-γ水平;蛋白印迹法分析各组p-Drp1、MnSOD蛋白表达;DHE染色及流式检测各组ROS水平;激光共聚焦观察各组线粒体形态。结果ELISA显示,与对照组比较,TSLP组IL-4、IL-5水平明显升高,IFN-γ水平下调(P<0.05);与TSLP组比较,5、10μmol·L^-1 Mdivi-1组IL-4、IL-5水平降低,10μmol·L^-1 Mdivi-1组IFN-γ浓度有所回升(P<0.05)。DHE染色及流式显示,TSLP组ROS水平较对照组升高;而5、10μmol·L^-1 Mdivi-1组ROS水平较TSLP组降低(P<0.05)。共聚焦显示,TSLP刺激后,细胞内大量圆球状线粒体生成,5、10μmol·L^-1 Mdivi-1干预后该现象得到改善。蛋白印迹显示,TSLP组p-Drp1水平明显上调,MnSOD表达下降,Mdivi-1干预则有效逆转了上述变化。结论线粒体动态失衡可能是NK细胞异常活化内在机制之一,其或是调控NK细胞NK2型活化介导的变应性炎症反应的重要靶点。

Study on effect of restoring mitochondrial dynamics on NK2 activation of NK cells

Aim To explore the internal mechanism of NK2 activation of NK cells from the perspective of“mitochondrial dysfunction-abnormal cell activation”.Methods NK-92MI cells were divided into blank group,TSLP group,1,5,and 10μmol·L^-1 Mdivi-1 dose groups.The levels of IL-4,IL-5,and IFN-γin the supernatant of each group were determined by ELISA;The expression of p-Drp1 and MnSOD protein in each group was determined by Western blotting;the ROS level of each group was detected by DHE staining and flow cytometry;mitochondrial morphology was observed by confocal laser in each group of cells.Results ELISA showed that compared with control group,the levels of IL-4 and IL-5 in cell supernatant of TSLP group significantly increased,and the level of IFN-γwas down-regulated(P<0.05);Compared with TSLP group,the levels of IL-4 and IL-5 in cell supernatant of 5 and 10μmol·L^-1 Mdivi-1 group decreased,and the IFN-γconcentration of the 10μmol·L^-1 Mdivi-1 group rose(P<0.05).DHE staining and flow cytometry showed that ROS level of cells in TSLP group was significantly higher than control group.Compared with TSLP group,ROS level of the 5 and 10μmol·L^-1 Mdivi-1 groups decreased(P<0.05).The laser confocal results showed that after TSLP stimulation,a large number of spherical mitochondria were formed in cells.This phenomenon was improved to a certain extent after the intervention of 5,10μmol·L^-1 Mdivi-1.Western blot analysis showed that the p-Drp1 level of NK-92MI cells in TSLP group was significantly up-regulated,and the expression of MnSOD decreased,while the intervention of Mdivi-1 effectively reversed the changes in the expression of the above-mentioned molecules.Conclusions Mitochondrial dynamic imbalance may be one of the internal mechanisms of abnormal activation of NK cells,and it may be an important target for regulating NK2 activation of NK cells and improving the allergic inflammatory response mediated by it.