线粒体DNA在百草枯中毒机制中的作用

摘要

目的 利用体外实验研究线粒体DNA (mtDNA)在百革枯(PQ)致病机理中的作用.方法 首先用PQ处理人Ⅱ型肺泡上皮细胞(A549细胞),应用CCK-8法检测细胞生存率,然后利用绝对定量PCR的方法检测培养上清液中mtDNA的含量,以激光共聚焦成像法检测线粒体膜电位来验证PQ对线粒体的损伤作用.接着通过Transwell趋化实验检测mtDNA对入外周血单个核细胞(PBMC)及中性粒细胞(PMN)的趋化活性,并用流式细胞术确定趋化细胞的亚型.同时应用Xcelligence系统和体外血管通透性试剂盒检测了mtDNA对血管通透性的影响.最后验证mtDNA在细胞增殖方面的作用.结果 PQ致A549细胞损伤的半数致死浓度为600 μmol/L,且A549细胞的生存率及对线粒体的损伤存在浓度和时间依赖关系(P＜0.05).PQ致细胞损伤释放出的mtDNA可以非特异地趋化单核细胞,但在血管通透性方面没有影响.并且mtDNA不能直接刺激人成纤维细胞的增殖,但可以引起主要的促纤维化因子TGF-β1的增加,与对照组相比,差异具有统计学意义(P＜0.05).结论 PQ损伤细胞释放的mtDNA在PQ所致炎症及增殖反应中起着重要的作用.%Objective To investigate the effects of mitochondrial DNA (mtDNA) on the pathopoiesis mechanisms of paraquat poisoning in vitro.Methods Firstly,the survival rate of A549 cells (human type Ⅱ alveolar epithelial cells) was measured with cell counting kit-8 after exposure to paraquat.Afterwards,the concentration of mtDNA in supernant of culture medium for culturing A549 and the chauge of mitochondrial membrance potential were detected with absolute quantitative PCR and confocal laser microscopy,respectively.Then,The chemotactic activity of mtDNA in peripheral blood mononuclear cells (PBMC) and neutrophils (PMN) were detected by transwell chemotaxis,and the subtype of chemotactic cells was measured with flow cytometry.Meanwhile,the role of mtDNA in vascular permeability was measured by using Xcelligence system and in vitro using vascular permeability kits.Finally,the effects of mtDNA in cell proliferation were to verify.Results The 50％ of lethal concentration (LD50) of paraquat for A549 was 600 μmol/L.Cell viability and concentration of mtDNA following challenge of PQ revealed in a concentration-and time-dependent manner (P ＜ 0.05).The mtDNA had a power in aggregating PBMC nonspecifically,but there was no effect on the vascular permeability was found.Moreover,the proliferation of human fibroblasts was not stimulated directly by mtDNA,but TGF-β1 (transforming growth factor-beta 1),a major pro-fibrotic factor,was increased compared to control group (P ＜ 0.05).Conclusions The mtDNA could play an important role in the inflammatory and proliferation responses to paraquat poisoning.