TIPE2负向调控RNA病毒感染效应及其机制研究

发布时间：2018-02-17 04:49

本文关键词： RNA病毒负向调控信号通路肿瘤坏死因子α诱导蛋白8样分子2　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景及目的:急性呼吸道感染是儿童感染性疾病中最常见的类型,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病毒感染尚无有效的治疗药物,所以探索RNA病毒感染的调控机制,可以为寻求治疗RNA病毒感染的新方法提供理论依据和研究目标。肿瘤坏死因子α诱导蛋白8样分子2(Tumor necrosis factor-α-induced protein 8-like 2,TIPE2)是一个最近发现的免疫负调控因子,在先天性免疫应答和获得性免疫应答中均起重要的负向调控作用。然而TIPE2对RNA病毒感染的调节,尤其是TIPE2对RNA病毒感染机体的的信号途径的调控,及TIPE2如何调控信号途径尚未有报道。本研究的目的是探讨TIPE2对RNA病毒感染的调控作用及其相关分子机制。方法:1.以2014年11月至2016年10月因临床确诊呼吸道合胞病毒(Respiratory syncytial virus,RSV)(儿童最常见的RNA病毒)感染在我院治疗的患儿154例及66例健康体健儿童为研究对象,检测外周血单个核细胞中TIPE2表达的变化。2.同时在体外细胞水平检测了水疱性口炎病毒(Vesicular Stomatitis Virus,VSV)(科研常用的RNA病毒)和RSV感染后巨噬细胞(鼠原代腹腔巨噬细胞、鼠巨噬细胞Raw264.7、经诱导人单核细胞THP-1、经诱导人外周血单核细胞)TIPE2 mRNA和蛋白表达水平的影响。3.通过干扰和过表达实验研究TIPE2对RNA病毒感染的调控作用,通过Western-blot实验探索其如何调控RNA病毒感染巨噬细胞后信号通路的活化情况。结果:1.在临床标本实验中,我们发现RSV感染患儿外周血单核细胞中TIPE2 mRNA表达较健康儿童下调。2.在细胞实验中进一步证实VSV感染原代腹腔巨噬细胞后,TIPE2的表达(包括mRNA和蛋白水平)均可明显下调。VSV感染Raw264.7细胞和THP-1细胞同样可以降低TIPE2的表达。另一种RNA病毒RSV刺激腹腔巨噬细胞TIPE2表达同样下调。3.干扰了巨噬细胞内源性TIPE2后,被VSV感染的巨噬细胞数明显减少,VSV-G基因的表达量也显著下调。而在巨噬细胞内过表达TIPE2能够显著的促进VSV的复制和感染。4.干扰TIPE2的巨噬细胞在RNA病毒感染下能够显著促进I型干扰素和炎性因子的分泌,而过表达TIPE2,则能够显著抑制I型千扰素和炎性因子的分泌。5.TIPE2能够通过抑制TBK1、IRF3信号途径的活化,抑制I型干扰素和炎性因子的分泌。结论:1.在临床标本实验中证实了 RSV(RNA病毒)感染后TIPE2表达下调。2.在细胞实验水平中进一步证实了 RNA病毒感染后TIPE2表达下调。3.TIPE2能够促进RNA病毒的感染与复制。4.TIPE2能够抑制由RNA病毒感染诱导的I型干扰素和炎性细胞因子的产生。5.TIPE2可能通过抑制TBK1、IRF3信号通路的活化来抑制由RNA病毒感染诱导的I型干扰素和炎性细胞因子的产生,促进RNA病毒的感染与复制。
[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- 伪 -inducible protein 8-like molecule 2Tumor necrosis factor- 伪 -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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R725.1

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