Nur77在LPS诱导下对小胶质细胞激活的蛋白质组学分析

发布时间：2018-01-05 11:05

本文关键词：Nur77在LPS诱导下对小胶质细胞激活的蛋白质组学分析　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

更多相关文章： 小胶质细胞 LPS 质谱分析 信号通路

【摘要】：[目的]小胶质细胞是中枢神经系统的固有免疫细胞,在中枢神经系统免疫反应中起着重要作用。Nur77在中枢神经系统内高表达,参与炎症反应及细胞增殖等生物进程。本实验的研究旨在探讨Nur77在LPS诱导的小胶质细胞激活中的作用。[方法]用LPS刺激WT及Nur77-/-原代小胶质细胞,收集细胞蛋白并进行质谱分析。用实时定量PCR(Q-PCR)及免疫蛋白印记(western blot)实验的方法验证差异性蛋白的表达。[结果]在WT及Nur77-/-小胶质细胞中,共有2004种差异性蛋白,其中在LPS刺激前后分别有749及677种蛋白差异性表达。Nur77-/-小胶质细胞在LPS刺激后差异性蛋白基因本体学分析。在细胞组分方面上调蛋白包括核糖体、核糖核蛋白复合物及胞质小核糖体亚基等,下调蛋白包括蛋白酶体、蛋白酶体调节微粒及蛋白酶体附件复合物等;在分子功能方面上调蛋白涉及氨基肽酶的调节活化、肝糖原的磷酸化激活及磷酸化酶的活化等,下调蛋白涉及参与核小体的DNA结合,突触融合蛋白的结合及SNAP受体活化等;在生物学功能方面上调蛋白可参与蛋白转运、跨膜蛋白转运及囊泡转运等功能,下调蛋白主要参与囊泡储存,囊泡溶解及跨膜转运等功能。对差异蛋白进行信号通路分析显示LPS后Nur77-/-小胶质细胞的差异性蛋白分布在小胶质细胞激活的重要信号通路中,其中包括Toll样受体信号通路、MAPK信号通路、FcyR调节的吞噬作用及小胶质细胞的催化作用。此外通过对差异蛋白的分析我们发现Nur77可能是vav1及ERK1/2信号通路中的上游物质。[结论]本篇研究主要是研究Nur77敲除小胶质细胞中蛋白表达水平的变化并且初步探索了LPS后Nur77对小胶质细胞的活化作用。LPS后Nur77可影响小胶质细胞激活的信号通路,其中包括Toll样受体信号通路、MAPK信号通路、FcγR调节的吞噬作用及趋化相关信号通路。同时,我们也首次发现Nur77可通过ERK1/2信号通路及Vav1的表达来调节LPS诱导的小胶质细胞的激活。为研究Nur77在LPS诱导的小胶质细胞激活的作用提供了新的研究视角。
[Abstract]:[Objective] microglia are innate immune cells of the central nervous system, which play an important role in the immune response of the central nervous system. Nur77 is highly expressed in the central nervous system. The aim of this study was to investigate the role of Nur77 in the activation of microglia induced by LPS. [Methods] WT and Nur77-r-primary microglia were stimulated with LPS. Cell proteins were collected and analyzed by mass spectrometry. The expression of differentially expressed proteins was verified by real-time quantitative PCRQ-PCR and Western blot assay. [Results] in WT and Nur77-r-microglia, there were four hundred and four different proteins. Of these, 749 and 677 proteins were differentially expressed before and after LPS stimulation. Nur77-r.-differential protein gene ontological analysis of microglial cells after LPS stimulation. Modulins include ribosomes. Ribonucleoprotein complexes and cytoplasmic small ribosomal subunits, down-regulated proteins include proteasome, proteasome regulatory particles and proteasome adnexal complexes. The up-regulation of protein involves the regulation of aminopeptidase, the phosphorylation of liver glycogen and the activation of phosphorylase, and the down-regulation of protein is involved in the DNA binding of nucleosome. Synaptic fusion protein binding and SNAP receptor activation; In biological function, up-regulation of protein involved in protein transport, transmembrane protein transport and vesicle transport, and down-regulation of protein mainly involved in vesicle storage. The signal pathway analysis of differential proteins showed that the differential proteins of Nur77-r-microglia were distributed in the important signal pathway activated by microglia after LPS. These include Toll like receptor signaling pathway and MAPK signal pathway. FcyR regulates phagocytosis and microglia catalyze. In addition, we found that Nur77 may be the upstream substance in vav1 and ERK1/2 signaling pathway by analyzing differential proteins. [Conclusion: this study is mainly to study the changes of protein expression in Nur77 knockout microglia and to explore the activation of Nur77 on microglia after LPS. 7can affect the signal pathway activated by microglia. These include phagocytosis of FC 纬 R regulation and chemoattractant signal pathway in Toll like receptor signaling pathway. We also found for the first time that Nur77 can regulate the activation of LPS induced microglia by ERK1/2 signaling pathway and Vav1 expression. The role of cellular activation provides a new perspective for research.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R741

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