本文關(guān)鍵詞： RNA病毒 負(fù)向調(diào)控 信號(hào)通路 腫瘤壞死因子α誘導(dǎo)蛋白8樣分子2　出處：《浙江大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研究背景及目的:急性呼吸道感染是兒童感染性疾病中最常見(jiàn)的類(lèi)型,90%由病毒感染引起,而病毒中以RNA病毒居多。目前除了流感病毒外,其他RNA病毒感染尚無(wú)有效的治療藥物,所以探索RNA病毒感染的調(diào)控機(jī)制,可以為尋求治療RNA病毒感染的新方法提供理論依據(jù)和研究目標(biāo)。腫瘤壞死因子α誘導(dǎo)蛋白8樣分子2(Tumor necrosis factor-α-induced protein 8-like 2,TIPE2)是一個(gè)最近發(fā)現(xiàn)的免疫負(fù)調(diào)控因子,在先天性免疫應(yīng)答和獲得性免疫應(yīng)答中均起重要的負(fù)向調(diào)控作用。然而TIPE2對(duì)RNA病毒感染的調(diào)節(jié),尤其是TIPE2對(duì)RNA病毒感染機(jī)體的的信號(hào)途徑的調(diào)控,及TIPE2如何調(diào)控信號(hào)途徑尚未有報(bào)道。本研究的目的是探討TIPE2對(duì)RNA病毒感染的調(diào)控作用及其相關(guān)分子機(jī)制。方法:1.以2014年11月至2016年10月因臨床確診呼吸道合胞病毒(Respiratory syncytial virus,RSV)(兒童最常見(jiàn)的RNA病毒)感染在我院治療的患兒154例及66例健康體健兒童為研究對(duì)象,檢測(cè)外周血單個(gè)核細(xì)胞中TIPE2表達(dá)的變化。2.同時(shí)在體外細(xì)胞水平檢測(cè)了水皰性口炎病毒(Vesicular Stomatitis Virus,VSV)(科研常用的RNA病毒)和RSV感染后巨噬細(xì)胞(鼠原代腹腔巨噬細(xì)胞、鼠巨噬細(xì)胞Raw264.7、經(jīng)誘導(dǎo)人單核細(xì)胞THP-1、經(jīng)誘導(dǎo)人外周血單核細(xì)胞)TIPE2 mRNA和蛋白表達(dá)水平的影響。3.通過(guò)干擾和過(guò)表達(dá)實(shí)驗(yàn)研究TIPE2對(duì)RNA病毒感染的調(diào)控作用,通過(guò)Western-blot實(shí)驗(yàn)探索其如何調(diào)控RNA病毒感染巨噬細(xì)胞后信號(hào)通路的活化情況。結(jié)果:1.在臨床標(biāo)本實(shí)驗(yàn)中,我們發(fā)現(xiàn)RSV感染患兒外周血單核細(xì)胞中TIPE2 mRNA表達(dá)較健康兒童下調(diào)。2.在細(xì)胞實(shí)驗(yàn)中進(jìn)一步證實(shí)VSV感染原代腹腔巨噬細(xì)胞后,TIPE2的表達(dá)(包括mRNA和蛋白水平)均可明顯下調(diào)。VSV感染Raw264.7細(xì)胞和THP-1細(xì)胞同樣可以降低TIPE2的表達(dá)。另一種RNA病毒RSV刺激腹腔巨噬細(xì)胞TIPE2表達(dá)同樣下調(diào)。3.干擾了巨噬細(xì)胞內(nèi)源性TIPE2后,被VSV感染的巨噬細(xì)胞數(shù)明顯減少,VSV-G基因的表達(dá)量也顯著下調(diào)。而在巨噬細(xì)胞內(nèi)過(guò)表達(dá)TIPE2能夠顯著的促進(jìn)VSV的復(fù)制和感染。4.干擾TIPE2的巨噬細(xì)胞在RNA病毒感染下能夠顯著促進(jìn)I型干擾素和炎性因子的分泌,而過(guò)表達(dá)TIPE2,則能夠顯著抑制I型千擾素和炎性因子的分泌。5.TIPE2能夠通過(guò)抑制TBK1、IRF3信號(hào)途徑的活化,抑制I型干擾素和炎性因子的分泌。結(jié)論:1.在臨床標(biāo)本實(shí)驗(yàn)中證實(shí)了 RSV(RNA病毒)感染后TIPE2表達(dá)下調(diào)。2.在細(xì)胞實(shí)驗(yàn)水平中進(jìn)一步證實(shí)了 RNA病毒感染后TIPE2表達(dá)下調(diào)。3.TIPE2能夠促進(jìn)RNA病毒的感染與復(fù)制。4.TIPE2能夠抑制由RNA病毒感染誘導(dǎo)的I型干擾素和炎性細(xì)胞因子的產(chǎn)生。5.TIPE2可能通過(guò)抑制TBK1、IRF3信號(hào)通路的活化來(lái)抑制由RNA病毒感染誘導(dǎo)的I型干擾素和炎性細(xì)胞因子的產(chǎn)生,促進(jìn)RNA病毒的感染與復(fù)制。
[Abstract]:Background and objective: acute respiratory tract infection (ARI) is the most common type of infectious disease in children, 90% of which are caused by virus infection, and RNA virus is the most common virus. At present, there is no effective therapy for RNA virus infection except influenza virus. So to explore the regulatory mechanism of RNA virus infection, TNF- 偽 -inducible protein 8-like molecule 2Tumor necrosis factor- 偽 -induced protein 8-like TIPE2) is a recently discovered negative immunomodulatory factor. Both innate and acquired immune responses play an important role in negative regulation. However, the regulation of RNA infection by TIPE2, especially the regulation of signal pathway of RNA virus infection by TIPE2, may play an important role in both innate and acquired immune responses. The purpose of this study was to investigate the regulatory effect of TIPE2 on RNA virus infection and its related molecular mechanisms. Methods: 1. From November 2014 to October 2016, the clinical diagnosis of respiratory syncytial disease was carried out. 154 children with respiratory syncytial virus (RSVV) infection and 66 healthy children were studied. The changes of TIPE2 expression in peripheral blood mononuclear cells (PBMC) were detected. At the same time, vesicular Stomatitis virus (Vesicular Stomatitis virus) and macrophages (murine primary peritoneal macrophages) after RSV infection were detected at the cell level. Murine macrophage Raw264.7, human monocyte THP-1, and human peripheral blood monocytes induced TIPE2 mRNA and protein expression levels. 3. To investigate the regulatory effect of TIPE2 on RNA virus infection by interfering and overexpression experiments. Western-blot experiment was used to explore how to regulate the activation of signal pathway after RNA virus infection in macrophages. Results: 1. We found that the expression of TIPE2 mRNA in peripheral blood monocytes of children with RSV infection was lower than that of healthy children. In cell experiment, it was further confirmed that the expression of TIPE2 (including mRNA and protein levels) in primary peritoneal macrophages infected with VSV was significant. Down-regulation of TIPE2 expression in Raw264.7 cells and THP-1 cells induced by RNA virus RSV also down-regulated TIPE2 expression in peritoneal macrophages, which interfered with endogenous TIPE2 in macrophages. The number of macrophages infected with VSV significantly decreased the expression of VSV-G gene, while overexpression of TIPE2 in macrophages significantly promoted the replication and infection of VSV. 4. Macrophages that interfered with TIPE2 could be infected with RNA virus. The secretion of interferon type I and inflammatory factors was significantly increased. However, overexpression of TIPE2 could significantly inhibit the secretion of type I and inflammatory cytokines. 5. TIPE2 could inhibit the activation of TBK1 and IRF3 signaling pathway. Inhibition of the secretion of interferon type I and inflammatory cytokines. Conclusion 1. The down-regulation of TIPE2 expression after infection of RSV(RNA virus was confirmed in clinical specimens. 2. The down-regulation of TIPE2 expression after infection of RNA virus was further confirmed at the cell level. 3.TIPE2 can promote the infection and replication of RNA virus. 4. TIPE2 can inhibit the production of type I interferon and inflammatory cytokines induced by RNA virus infection. 5. TIPE2 may inhibit the activation of TBK1 / IRF3 signaling pathway and inhibit the production of TBK1- IRF3 signaling pathway induced by RNA virus infection. The production of interferon type I and inflammatory cytokines, To promote the infection and replication of RNA virus.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R725.1

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