CCCP对人外周血CD4^+CD25^+调节性T细胞线粒体自噬的影响探讨

The Effect of CCCP on Mitochondrial Autophagy of CD4^+CD25^+ Regulatory T Cells in Human Peripheral Blood

目的:探讨不同浓度羰基氰化物间氯苯腙(carbonyl cyanide m-chlorophenylhydrazone,CCCP)对人外周血调节性T细胞(regulatory T cell,Tregs)线粒体自噬的影响及作用机制。方法:采集健康成年人的外周血,用Ficoll密度梯度离心法分离出人外周血单个核细胞,选用基于分离柱的人CD4+CD25+调节性T细胞分选试剂盒分离得到Tregs,对Tregs鉴定后分组,分别用浓度为2.5μmol/L(C组)、5.0μmol/L(D组)、10.0μmol/L(E组)、20.0μmol/L(F组)、40.0μmol/L(G组)的CCCP作用24 h,流式细胞仪检测各组Tregs的活性氧(reactive oxygen species,ROS)水平和线粒体膜电位,荧光显微镜观察各组Tregs线粒体和溶酶体的共定位情况,流式细胞仪检测各组Tregs的线粒体质量。结果:磁珠分选得到的CD4+CD25+Treg细胞经抗体标记后鉴定,纯度在(93. 36±1. 87)%之间;随着CCCP作用浓度逐渐变大,ROS的水平逐渐升高,线粒体膜电位逐渐降低(P<0.05);从线粒体和溶酶体融合情况看:当CCCP的作用浓度在5～10μmol/L时,橙色(黄色)荧光水平最强,随着CCCP浓度继续升高,橙色(黄色)荧光水平下降(P<0.05);线粒体质量随着CCCP作用浓度的增加而下降,组间比较发现,C、D、E组之间无明显差异,与E组相比,F和G组线粒体质量明显下降,P<0.05。结论:随着CCCP作用浓度(0～10μmol/L)的逐渐变大,Treg细胞内的ROS水平升高,线粒体膜电位降低,促进线粒体自噬,线粒体质量保持动态平衡;当ROS堆积,线粒体膜电位明显下降时,线粒体自噬被抑制,线粒体质量受损。

Objective To investigate the effects and mechanisms of different concentrations of carbonyl cyanide m-chlorophenylhydrazone(CCCP)on mitochondrial autophagy of regulatory T cells(Tregs)in human peripheral blood.Methods Peripheral blood of healthy adults was collected and human peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation.Tregs were separated using human CD4+CD25+regulatory T cell sorting kit.Tregs were identified and grouped.Tregs'mitochondrial membrane potential(MMP)and reactive oxygen species(ROS)levels were measured by flow cytometry and the co-localization of mitochondria and lysosomes was observed by fluorescence microscopy after 24 hours of CCCP treatment with concentrations of 2.5μmol/L,5.0μmol/L,10.0μmol/L,20.0μmol/L and 40.0μmol/L respectively.Flow cytometry was used to detect the mitochondrial mass of Tregs.Results The purity of CD4+CD25+Treg cells sorted by magnetic beads and identified by antibody labeling was 93.36+1.87%.With the increasing concentration of CCCP,the level of ROS increased and MMP decreased gradually(P<0.05).The orange(yellow)fluorescence level,which represents the fusion of mitochondria and lysosomes,was the highest when the concentration of CCCP was 5~10μmol/L and decreased with the increase of CCCP concentration(P<0.05).The mitochondrial mass decreased with the increase of CCCP concentration.There was no significant difference among groups C,D and E.Compared with group E,the mitochondrial mass of group F and G decreased significantly(P<0.05).Conclusion As the concentration of CCCP(0~10μmol/L)gradually increased,the level of ROS in Treg cells increased,MMP decreased,mitochondrial autophagy was promoted,and mitochondrial mass remained dynamically balanced.When ROS accumulated,MMP decreased significantly,mitochondrial autophagy was inhibited and mitochondrial integrity was impaired.