鸟结核分枝杆菌感染巨噬细胞后的外泌体刺激巨噬细胞产生的细胞因子及其蛋白质表达改变  

Changes of cytokines and protein expression of macrophages stimulated with exosome from macrophages by Mycobacterium avium infection

作　　者：王建军[1,2] 陈偲[1] 谢萍芳[1] 潘艺[1] 谭云洪 汤立军[1,4] 

机构地区：[1]中南大学生物科学与技术学院分子生物学研究中心,湖南长沙410078 [2]江苏省昆山市第一人民医院,江苏昆山215300 [3]湖南省结核病防治所,湖南长沙410205 [4]中南大学生物科学与技术学院生物实验室,湖南长沙410013

出　　处：《细胞与分子免疫学杂志》2013年第2期123-126,131,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81071326);湖南省杰出青年基金(04JJ1006)

摘　　要：目的探讨鸟结核分枝杆菌(M.avium)感染巨噬细胞后分泌的外泌体[(+)外泌体]刺激巨噬细胞后产生的分子免疫机制及其差异蛋白质成分研究。方法冷冻高速离心法收集经鸟分枝结核杆菌感染及未感染的巨噬细胞上清中的外泌体,然后利用外泌体刺激巨噬细胞;ELISA方法检测经外泌体刺激巨噬细胞后细胞上清中IFN-γ、TNF-α的含量;流式细胞术从蛋白水平检测巨噬细胞受外泌体刺激后CD80、CD86蛋白表达情况;2-DE MALDI-TOF/TOF MS技术分析鉴定巨噬细胞受M.avium感染后分泌的外泌体中的差异蛋白。结果 IFN-γ、TNF-α在(+)外泌体刺激巨噬细胞后培养上清中含量增加;CD80、CD86蛋白在巨噬细胞受(+)外泌体刺激后表达上调。外泌体经2-DE MALDI TOF/TOF MS分析获得18个差异蛋白,且质谱成功鉴定12个。结论 (+)外泌体促进巨噬细胞TNF-α、IFN-γ的分泌从而增强巨噬细胞的炎症反应,并且可促进巨噬细胞CD80、CD86蛋白表达增强;筛选出差异蛋白的功能与细胞骨架结构、蛋白质合成与加工,炎症反应密切相关。Objective To explore the mechanism underlying the molecular immune response of macrophages stimulated with exosome [（＋）exosome] from macrophages after Mycobacterium avium（M.avium） infection and analyze the differential protein component of the exosome.Methods The culture supernatants of M.avium-infected macrophages and uninfected ones were collected and exosome was harvested from the supernatants by frozen ultra centrifugation.The concentrations of IFN-γ,TNF-α in supernatants were detected by enzyme-linked immunosorbent assay（ELISA）,and CD80,CD86 expressions on macrophages were analyzed by flow cytometry after macrophages were stimulated with exosome.Meanwhile,2-DE MALDI TOF/TOF MS was used to identify differentially expressed proteins of exosome between M.avium infected group and uninfected group.Results IFN-γ,TNF-α concentrations were increased in the supernatant after stimulated with（＋）exosome and CD80,CD86 were raised on macrophage surface by stimulation with（＋）exosome.With 2-DE MALDI TOF/TOF MS analysis,we obtained 18 differentially expressed proteins and 12 proteins were identified successfully.Conclusion（＋）exosome induces TNF-α,IFN-γ secretion from macrophages and result in the promotion of inflammatory response.In addition,（＋）exosome enhances CD80 and CD86 protein expressions.The function of the differentially expressed proteins we identified is closely related to cytoskeleton,protein synthesis and processing,inflammatory response.

关 键 词：巨噬细胞 外泌体 M AVIUM 2-DE MALDI-TOF TOFMS 

分 类 号：R392.11[医药卫生—免疫学]