主要穹窿蛋白调控dsRNA和流感病毒激活的炎症通路及机制研究

发布时间：2018-03-12 10:48

  本文选题：主要穹窿蛋白　切入点：白细胞介素6　出处：《武汉大学》2016年博士论文　论文类型：学位论文

【摘要】：病原体感染机体以后,会诱导机体产生大量的细胞因子如干扰素,炎症因子和趋化因子,从而建立体内的抗病原体微环境,达到清除病原体,抑制病原体进一步损伤机体的目的。在我们原来细胞水平研究中,我们发现MVP与天然免疫反应中Ⅰ型干扰素的产生有关,丙型肝炎病毒(HCV)感染能够诱导机体产生主要穹窿蛋白(MVP),后者通过激活干扰素α大量表达,从而达到抑制HCV复制的目的；乙型肝炎病毒(HBV)也能够诱导MVP的产生,并且通过阻断MVP对MyD88介导的Ⅰ型干扰素通路的调控而实现免疫逃逸。但是,目前为止,尚无文献阐明MVP与病原体引发的炎症反应之间的关系。在本研究中,我们发现dsRNA类似物polyI:C以及甲型流感病毒(IAV)分别能够诱导人外周血淋巴细胞(PBMC)和A549细胞产生MVP,炎症因子白细胞介素6(IL6)以及趋化因子白细胞介素8(IL8),利用瞬时转染shMVP的表达质粒敲减内源MVP后,IL6和IL8的表达水平降低,因此我们初步推测MVP可能调控病原体诱导的炎症反应。通过基于pLKO.1-MVP shRNA慢病毒系统,我们构建了MVP稳定敲减的A549细胞系和THP.1细胞系,并且发现在两种MVP敲减的细胞系中,polyI:C诱导的IL6,IL8的表达水平受损,此外,MVP小鼠来源的脾脏细胞,腹腔巨噬细胞以及PBMC中,polyI:C以及IAV诱导产生的IL6和1L8表达水平显著低于野生型小鼠细胞。在揭示MVP调控炎症反应的背后机制过程中,我们利用荧光素酶报告基因实验发现,MVP能够显著上调IL6,IL8启动子活性,因此我们推测MVP在转录水平调控IL6和IL8的表达；此外,MVP能够协同转录因子c-Fos,C/EBPβ-LAP以及NF-KB激活IL6和IL8启动子,而在IL6和IL8启动子区域引入AP-1和C/EBPβ突变后,MVP对IL6和IL8启动子的激活失效。通过免疫共沉淀实验发现,polyI:C以及IAV刺激诱导MVP与c-Fos,C/EBPp发生相互作用,免疫荧光结果也显示MVP 与 c-Fos,C/EBPβ在polyl:C以及1AV处理的A549细胞内共定位。在探究MVP与转录因子相互作用后的下游事件的过程中,我们通过核质抽捉实验以及免疫荧光实验发现polyl:C以及IAV刺激MVP入核；染色质免疫共沉淀实验显示,MVP过表达能够增强转录因子c-Fos,C/EBPβ在IL6和IL8启动子区域的募集,MVP稳定敲减的A549细胞中,c-Fos,C/EBPβ在IL6和IL8启动子区域结合显著减少。在MVP-/-小鼠模型中,我们通过鼻腔滴注鼠适应性1AV/FM/1/47(H1N1)感染野生型以及MVP以小鼠,发现MVP“小鼠有更高的死亡率,在探究其更易感H1N1背后机制的过程中发现,病毒感染2天后,MVP-/-小鼠肺部早期抗病毒细胞因子mIL6,mCxcl1,mCxcl2,mCxcl5,mIL1-β,mTNF-α和mIFN-α表达受损,导致肺部更高滴度H1N1,通过分析小鼠肺部病理切片发现,H1N1定位于鼠肺部气管柱状上皮细胞,高滴度H1N1病毒引起气管上皮细胞更严重的坏死和脱落,最终导致MVP-/-小鼠低生存率本论文研究结果首次通过体外细胞实验以及MVP-/-小鼠体内实验,揭示了MVP在炎症反应中的角色,并阐明背后机制：MVP在病原体诱导下,与转录因子c-Fos,C/EBPβ相互作用,促进转录因子入核并结合在下游细胞因子1L6和IL8启动子区域,激活IL6和IL8启动子活性,增强IL6和IL8的表达。其为更进-步的阐明MVP在病原体引起的天然免疫反应中提供了新的理论信息。
[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.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392
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本文编号：1601224