脂多糖诱导小鼠腹腔巨噬细胞parkin表达及线粒体自噬形成  

Lipopolysaccharide induces parkin expression and mitophagy in murine peritoneal macrophages

作　　者：程艳伟[1] 靳梦醒 闫海[1] 黄大可[2] 黄保军[1] 张林杰[1] 

机构地区：[1]安徽医科大学基础医学院免疫学教研室,合肥230032 [2]安徽医科大学基础医学院综合实验室,合肥230032

出　　处：《中国免疫学杂志》2014年第11期1457-1461,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金(30972791)资助

摘　　要：目的:探讨巨噬细胞在脂多糖(LPS)处理后parkin表达及对线粒体自噬的影响。方法:无菌分离并原代培养小鼠腹腔巨噬细胞;200 ng/ml LPS作用巨噬细胞,JC-1标记线粒体,流式细胞仪检测线粒体膜电位变化;RT-PCR检测巨噬细胞parkin mRNA水平;Western blot检测parkin及自噬相关蛋白LC3Ⅱ/LC3Ⅰ的表达水平;激光共聚焦显微镜分别检测巨噬细胞在LPS处理前后parkin在细胞内的分布及与LC3和线粒体三者的共定位情况。结果:JC-1染色流式细胞仪检测结果显示,LPS处理后,巨噬细胞线粒体膜电位降低,并随LPS作用时间延长呈持续下降趋势;parkin mRNA在LPS处理6 h内仅有少量的增加,但parkin蛋白水平在LPS刺激6 h后增加明显;parkin在巨噬细胞定位结果表明,未受LPS处理时parkin呈弥散均匀分布,而LPS刺激后parkin呈明显的点状聚集;Western blot检测结果显示巨噬细胞在LPS刺激后,LC3Ⅱ/LC3Ⅰ比例增大,表明巨噬细胞发生明显的自噬;激光共聚焦显微镜结果显示,巨噬细胞在LPS刺激后,parkin、LC3和线粒体三者存在共定位关系。结论:巨噬细胞在LPS刺激后,parkin表达增加并介导线粒体自噬形成,参与对损伤线粒体的清除,可能在巨噬细胞参与的炎症反应中发挥一定的调控作用。Objective： To investigate whether lipopolysaccharide induced parkin expression and mitophagy in macrophages. Methods： The murine peritoneal primary macrophages were aseptically isolated from Kunming mice and cultured in complete medium. The mitochondrial membrane potential of macrophages was detected by flow cytometry,after the cells were stimulated with200 ng /ml LPS and labeled mitochondria with JC-1. The parkin mRNA level of macrophages was detected by RT-PCR,protein levels of parkin and autophagic related protein LC3 Ⅱ and LC3 Ⅰ were determined by Western blot. The distribution and co-localization of parkin with LC3 and mitochondria in macrophages were respectively observed by laser scanning confocal microscope,before and after the cells were treated with LPS. Results： Flow cytometry results after JC-1 staining showed that mitochondrial membrane potential in macrophages was declined after stimulation with 200 ng /ml LPS,and continuously decreased with prolonged treatment time. The mRNA levels of parkin were increased slightly within 6 h after LPS stimulation,but parkin proteins were increased significantly within 6 h after LPS stimulation. The results of parkin distribution showed that parkin was evenly distributed in the cytoplasm at normal status,but became the obvious punctate distribution after LPS stimulation in macrophages. Western blot results showed LC3 Ⅱ /LC3 Ⅰ levels were increased after LPS stimulation,indicating the appearance of macrophage autophagy. Confocal microscopy showed that there were co-localization of parkin,LC3 and mitochondrial in macrophages after LPS stimulation. Conclusion： Parkin expression is increased significantly and mediated mitochondrial autophagy in macrophages after LPS stimulation,which is involved in the clearance of damaged mitochondria,thereby playing a role in regulating macrophage inflammatory response.

关 键 词：巨噬细胞 脂多糖 线粒体自噬 PARKIN 

分 类 号：R378.2[医药卫生—病原生物学] R392.1[医药卫生—基础医学]