本文選題：主要穹窿蛋白　切入點：白細胞介素6　出處：《武漢大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：病原體感染機體以后,會誘導(dǎo)機體產(chǎn)生大量的細胞因子如干擾素,炎癥因子和趨化因子,從而建立體內(nèi)的抗病原體微環(huán)境,達到清除病原體,抑制病原體進一步損傷機體的目的。在我們原來細胞水平研究中,我們發(fā)現(xiàn)MVP與天然免疫反應(yīng)中Ⅰ型干擾素的產(chǎn)生有關(guān),丙型肝炎病毒(HCV)感染能夠誘導(dǎo)機體產(chǎn)生主要穹窿蛋白(MVP),后者通過激活干擾素α大量表達,從而達到抑制HCV復(fù)制的目的；乙型肝炎病毒(HBV)也能夠誘導(dǎo)MVP的產(chǎn)生,并且通過阻斷MVP對MyD88介導(dǎo)的Ⅰ型干擾素通路的調(diào)控而實現(xiàn)免疫逃逸。但是,目前為止,尚無文獻闡明MVP與病原體引發(fā)的炎癥反應(yīng)之間的關(guān)系。在本研究中,我們發(fā)現(xiàn)dsRNA類似物polyI:C以及甲型流感病毒(IAV)分別能夠誘導(dǎo)人外周血淋巴細胞(PBMC)和A549細胞產(chǎn)生MVP,炎癥因子白細胞介素6(IL6)以及趨化因子白細胞介素8(IL8),利用瞬時轉(zhuǎn)染shMVP的表達質(zhì)粒敲減內(nèi)源MVP后,IL6和IL8的表達水平降低,因此我們初步推測MVP可能調(diào)控病原體誘導(dǎo)的炎癥反應(yīng)。通過基于pLKO.1-MVP shRNA慢病毒系統(tǒng),我們構(gòu)建了MVP穩(wěn)定敲減的A549細胞系和THP.1細胞系,并且發(fā)現(xiàn)在兩種MVP敲減的細胞系中,polyI:C誘導(dǎo)的IL6,IL8的表達水平受損,此外,MVP小鼠來源的脾臟細胞,腹腔巨噬細胞以及PBMC中,polyI:C以及IAV誘導(dǎo)產(chǎn)生的IL6和1L8表達水平顯著低于野生型小鼠細胞。在揭示MVP調(diào)控炎癥反應(yīng)的背后機制過程中,我們利用熒光素酶報告基因?qū)嶒灠l(fā)現(xiàn),MVP能夠顯著上調(diào)IL6,IL8啟動子活性,因此我們推測MVP在轉(zhuǎn)錄水平調(diào)控IL6和IL8的表達；此外,MVP能夠協(xié)同轉(zhuǎn)錄因子c-Fos,C/EBPβ-LAP以及NF-KB激活I(lǐng)L6和IL8啟動子,而在IL6和IL8啟動子區(qū)域引入AP-1和C/EBPβ突變后,MVP對IL6和IL8啟動子的激活失效。通過免疫共沉淀實驗發(fā)現(xiàn),polyI:C以及IAV刺激誘導(dǎo)MVP與c-Fos,C/EBPp發(fā)生相互作用,免疫熒光結(jié)果也顯示MVP 與 c-Fos,C/EBPβ在polyl:C以及1AV處理的A549細胞內(nèi)共定位。在探究MVP與轉(zhuǎn)錄因子相互作用后的下游事件的過程中,我們通過核質(zhì)抽捉實驗以及免疫熒光實驗發(fā)現(xiàn)polyl:C以及IAV刺激MVP入核；染色質(zhì)免疫共沉淀實驗顯示,MVP過表達能夠增強轉(zhuǎn)錄因子c-Fos,C/EBPβ在IL6和IL8啟動子區(qū)域的募集,MVP穩(wěn)定敲減的A549細胞中,c-Fos,C/EBPβ在IL6和IL8啟動子區(qū)域結(jié)合顯著減少。在MVP-/-小鼠模型中,我們通過鼻腔滴注鼠適應(yīng)性1AV/FM/1/47(H1N1)感染野生型以及MVP以小鼠,發(fā)現(xiàn)MVP“小鼠有更高的死亡率,在探究其更易感H1N1背后機制的過程中發(fā)現(xiàn),病毒感染2天后,MVP-/-小鼠肺部早期抗病毒細胞因子mIL6,mCxcl1,mCxcl2,mCxcl5,mIL1-β,mTNF-α和mIFN-α表達受損,導(dǎo)致肺部更高滴度H1N1,通過分析小鼠肺部病理切片發(fā)現(xiàn),H1N1定位于鼠肺部氣管柱狀上皮細胞,高滴度H1N1病毒引起氣管上皮細胞更嚴重的壞死和脫落,最終導(dǎo)致MVP-/-小鼠低生存率本論文研究結(jié)果首次通過體外細胞實驗以及MVP-/-小鼠體內(nèi)實驗,揭示了MVP在炎癥反應(yīng)中的角色,并闡明背后機制：MVP在病原體誘導(dǎo)下,與轉(zhuǎn)錄因子c-Fos,C/EBPβ相互作用,促進轉(zhuǎn)錄因子入核并結(jié)合在下游細胞因子1L6和IL8啟動子區(qū)域,激活I(lǐng)L6和IL8啟動子活性,增強IL6和IL8的表達。其為更進-步的闡明MVP在病原體引起的天然免疫反應(yīng)中提供了新的理論信息。
[Abstract]:The body after pathogen infection, will induce cells such as interferon cytokines, inflammatory cytokines and chemotactic factors, so as to establish the in vivo antiviral microenvironment, to remove pathogens, inhibit pathogen further injury to the body. In our original cell level study, we found that the production of type I MVP and innate immune response to interferon the hepatitis C virus (HCV) infection can induce major vault protein (MVP), the latter through activation of interferon alpha expression, so as to achieve the purpose of inhibiting HCV replication; hepatitis B virus (HBV) can induce the expression of MVP and MVP, by blocking the regulation of type I interferon pathway mediated by MyD88 the realization of immune escape. However, so far, there is no relationship between inflammation and MVP literature to clarify the pathogen. In this study, we found that dsR NA analogues polyI:C and influenza A virus (IAV) were able to induce human peripheral blood lymphocytes (PBMC) from MVP and A549 cells, inflammatory cytokines interleukin 6 (IL6) and chemokine interleukin 8 (IL8), the expression plasmid was transfected to shMVP knockdown of endogenous MVP. The expression level of IL6 and IL8 decreased, so we speculated that MVP may regulate pathogen induced inflammation. Through pLKO.1-MVP shRNA lentivirus based system, we construct a A549 cell line and THP.1 cell line MVP stable knockdown, and knockdown cell lines in two kinds of MVP, polyI:C induced IL6 expression. The level of IL8 damage, in addition, from MVP mice spleen cells and peritoneal macrophages and PBMC, the expression level of polyI:C and IAV induced IL6 and 1L8 cells was significantly lower than that of wild-type mice. In revealing the mechanism behind MVP regulate inflammation In the process, we use the luciferase reporter assay showed that MVP could significantly increase IL6 activity of IL8 promoter, so we speculate that the expression of MVP in transcriptional regulation of IL6 and IL8; in addition, MVP enhanced the transcription factor c-Fos, C/EBP -LAP and NF-KB beta activation of IL6 and IL8 promoter, and promoter region into AP-1 and C/EBP beta mutations in IL6 and IL8, MVP of IL6 and IL8 promoter activation failure. The experimental results showed that by immunoprecipitation, MVP and c-Fos and polyI:C induced by IAV stimulation, C/EBPp interaction, immunofluorescence results show that MVP and c-Fos, C/EBP and polyl:C in beta 1AV treated A549 cells co localization. In the process of exploring the downstream events of MVP and transcription factors interact in the US by pumping test and immunofluorescence and nuclear test showed that polyl:C and IAV stimulation of MVP into nucleus; chromatinimmune co precipitation experiment. Showed that overexpression of MVP can enhance the transcription factor c-Fos, beta C/EBP promoter region of IL6 and MVP raised in IL8 stable knockdown of A549 cells, c-Fos, C/EBP beta promoter region in IL6 and IL8 with significantly reduced. In a mouse model of MVP-/-, we through the nasal drip in the adaptability of 1AV/FM/1/47 (H1N1) the wild type and MVP infection in mice, MVP mice had a higher mortality rate, found in the process of exploring the mechanisms underlying H1N1 are more susceptible, infected 2 days later, MVP-/- in lungs of mice early antiviral cytokines mIL6, mCxcl1, mCxcl2, mCxcl5, mIL1- beta, impaired expression of mTNF- alpha and mIFN- alpha, cause the lungs more high titer of H1N1, through the analysis of mouse lung biopsy showed that H1N1 localized in the rat lung columnar epithelial cells, high titers of H1N1 virus caused by tracheal epithelial cells more severe necrosis and shedding, resulting in low survival rate of the mouse MVP-/- The results of this study for the first time through the experiments in vitro and in vivo MVP-/-, reveals the role of MVP in inflammation, and to elucidate the mechanism behind MVP in pathogen induced by c-Fos and transcription factor C/EBP beta interaction, promote nuclear transcription factor binding to the promoter region and downstream cytokine 1L6 and IL8 activation. The IL6 and IL8 promoter activity, enhanced expression of IL6 and IL8. It is more natural step in elucidating MVP immune response caused by pathogens in theory provides a new information.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R392
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本文編號：1601224