本文關(guān)鍵詞：Nur77在LPS誘導(dǎo)下對(duì)小膠質(zhì)細(xì)胞激活的蛋白質(zhì)組學(xué)分析　出處：《南京醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

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【摘要】：[目的]小膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)的固有免疫細(xì)胞,在中樞神經(jīng)系統(tǒng)免疫反應(yīng)中起著重要作用。Nur77在中樞神經(jīng)系統(tǒng)內(nèi)高表達(dá),參與炎癥反應(yīng)及細(xì)胞增殖等生物進(jìn)程。本實(shí)驗(yàn)的研究旨在探討Nur77在LPS誘導(dǎo)的小膠質(zhì)細(xì)胞激活中的作用。[方法]用LPS刺激WT及Nur77-/-原代小膠質(zhì)細(xì)胞,收集細(xì)胞蛋白并進(jìn)行質(zhì)譜分析。用實(shí)時(shí)定量PCR(Q-PCR)及免疫蛋白印記(western blot)實(shí)驗(yàn)的方法驗(yàn)證差異性蛋白的表達(dá)。[結(jié)果]在WT及Nur77-/-小膠質(zhì)細(xì)胞中,共有2004種差異性蛋白,其中在LPS刺激前后分別有749及677種蛋白差異性表達(dá)。Nur77-/-小膠質(zhì)細(xì)胞在LPS刺激后差異性蛋白基因本體學(xué)分析。在細(xì)胞組分方面上調(diào)蛋白包括核糖體、核糖核蛋白復(fù)合物及胞質(zhì)小核糖體亞基等,下調(diào)蛋白包括蛋白酶體、蛋白酶體調(diào)節(jié)微粒及蛋白酶體附件復(fù)合物等;在分子功能方面上調(diào)蛋白涉及氨基肽酶的調(diào)節(jié)活化、肝糖原的磷酸化激活及磷酸化酶的活化等,下調(diào)蛋白涉及參與核小體的DNA結(jié)合,突觸融合蛋白的結(jié)合及SNAP受體活化等;在生物學(xué)功能方面上調(diào)蛋白可參與蛋白轉(zhuǎn)運(yùn)、跨膜蛋白轉(zhuǎn)運(yùn)及囊泡轉(zhuǎn)運(yùn)等功能,下調(diào)蛋白主要參與囊泡儲(chǔ)存,囊泡溶解及跨膜轉(zhuǎn)運(yùn)等功能。對(duì)差異蛋白進(jìn)行信號(hào)通路分析顯示LPS后Nur77-/-小膠質(zhì)細(xì)胞的差異性蛋白分布在小膠質(zhì)細(xì)胞激活的重要信號(hào)通路中,其中包括Toll樣受體信號(hào)通路、MAPK信號(hào)通路、FcyR調(diào)節(jié)的吞噬作用及小膠質(zhì)細(xì)胞的催化作用。此外通過對(duì)差異蛋白的分析我們發(fā)現(xiàn)Nur77可能是vav1及ERK1/2信號(hào)通路中的上游物質(zhì)。[結(jié)論]本篇研究主要是研究Nur77敲除小膠質(zhì)細(xì)胞中蛋白表達(dá)水平的變化并且初步探索了LPS后Nur77對(duì)小膠質(zhì)細(xì)胞的活化作用。LPS后Nur77可影響小膠質(zhì)細(xì)胞激活的信號(hào)通路,其中包括Toll樣受體信號(hào)通路、MAPK信號(hào)通路、FcγR調(diào)節(jié)的吞噬作用及趨化相關(guān)信號(hào)通路。同時(shí),我們也首次發(fā)現(xiàn)Nur77可通過ERK1/2信號(hào)通路及Vav1的表達(dá)來調(diào)節(jié)LPS誘導(dǎo)的小膠質(zhì)細(xì)胞的激活。為研究Nur77在LPS誘導(dǎo)的小膠質(zhì)細(xì)胞激活的作用提供了新的研究視角。
[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 緯 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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R741

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本文編號(hào)：1382834