TLR3与RIG-I相关信号分子在鸭呼肠孤病毒感染雏鸭免疫器官中表达变化研究

发布时间：2018-04-01 20:36

本文选题：鸭呼肠孤病毒　切入点：TLR3　出处：《华中农业大学》2015年硕士论文

【摘要】：鸭呼肠孤病毒(Duck reovirus,DRV)感染雏鸭后,主要侵害雏鸭免疫系统,如脾、法氏囊、胸腺,引起雏鸭免疫器官损伤,从而继发其它细菌感染。天然免疫系统中有很多模式识别受体可以识别病毒核酸,从而激活机体的抗病毒反应。TLR3和RIG-I可以特异性的识别侵入机体的双链RNA病毒,激活下游的信号分子,最终诱导IFNβ的释放,引起抗病毒免疫反应。目前,对鸭呼肠孤病毒诱导抗病毒反应的信号通路还不明确,本研究通过病毒感染动物模型的复制,研究鸭呼肠孤病毒感染雏鸭后两条抗病毒反应信号通路中相关分子的m RNA表达变化,初步阐明病毒引起的主要抗病毒天然免疫信号途径。主要研究内容如下:1.鸭呼肠孤病毒感染雏鸭模型的复制将30羽樱桃谷雏鸭随机均分为感染组和对照组两组,于雏鸭7日龄对感染组雏鸭颈部皮下注射鸭呼肠孤病毒(HP20090318株)0.2m L/羽,对照组注射等量的生理盐水。分别于接毒后1d、3d、5d、7d、14d进行剖杀,每组剖杀3羽。眼观病理学观察,接毒后1d和3d雏鸭脾表面分布有大小不一的红色斑点,5d和7d雏鸭脾表面分布有灰白色病灶病灶周围有红色斑点;法氏囊与胸腺未见明显眼观病变。组织病变可见接毒后1d和3d脾组织内出现大量红细胞,红髓、白髓界限不清,接毒后5d和7d在组织内出现坏死灶及肉芽肿;感染组法氏囊淋巴滤泡数目减少,出现空泡;胸腺组织内未见明显病变。用免疫组化染色结果显示鸭呼肠孤病毒存在于脾巨噬细胞以及坏死灶中。RT-PCR检测鸭呼肠孤病毒S2基因,发现在接毒后1d、3d、5d、7d以及14d感染组雏鸭脾均有DRV存在。上述结果表明鸭呼肠孤病毒感染雏鸭模型复制成功。2.TLR3、TRIF、IRF3、RIG-I、IFNβ信号分子m RNA表达变化检测(1)TLR3抗病毒信号通路中,TLR3与其直接激活的TRIF在机体内表达水平在接毒后1d、3d、5d、7d以及14d表达差异不明显,在脾、胸腺与法氏囊三个组织中,法氏囊内各信号分子的表达水平高于脾和胸腺。IRF3是TLR3信号通路与RIG-I信号通路中共同的中间分子,其在脾与法氏囊中接毒后3d的相对表达水平与接毒后1d相比上升明显,接毒后5d、7d以及14d相对表达水平逐渐下降但与接毒后1d相比差异不明显,并且在不同感染日龄法氏囊内的相对表达水平均高于相应日龄的脾;IRF3在胸腺组织中接毒后1d表达明显上调,其他日龄上调不明显。(2)RIG-I抗病毒信号通路中RIG-I的表达水平要高于TLR3信号分子;其中在法氏囊内的表达水平高于脾和胸腺,在接毒后3d、5d表达水平最高。(3)IFNβ是两条信号通路的最终激活因子,并介导抗病毒效应,其在脾、胸腺与法氏囊三个组织中的相对表达水均呈现上调趋势,接毒后3d和5d相对表达水平上调。同样在法氏囊内的相对表达水平要高于脾和胸腺两组织。本研究结果表明:成功复制了鸭呼肠孤病毒HP20090318株感染雏鸭动物模型;鸭呼肠孤病毒进入雏鸭体内主要由RIG-I进行识别,为主要的抗病毒反应通路;在脾、法氏囊和胸腺三个组织中,法氏囊内各信号分子的表达水平要高于脾和胸腺,推测法氏囊在抗鸭呼肠孤病毒的作用中发挥着主要功能。
[Abstract]:Duck reovirus (Duck reovirus DRV) Infected Ducklings, mainly against duck immune system, such as the spleen, bursa, thymus, immune organs of ducks injury, thus secondary to other bacterial infections. The natural immune system has many pattern recognition receptors can recognize viral nucleic acid, double stranded RNA virus identification to activate antiviral the reaction of.TLR3 and RIG-I in the body can be specific to invade the body, the activation of downstream signaling molecules, induce IFN beta release, caused by antiviral immune response. At present, the signal pathway of duck reovirus induced antiviral response is not clear, the animal model of infection by the virus replication of duck reovirus Orphan Virus Infected Ducklings after M RNA two molecules related to antiviral response signal transduction in expression, preliminary clarify main antiviral virus induced innate immune signaling pathways. The main research contents Are as follows: 1. duck call model ducklings reovirus infection copy 30 broilers were randomly divided into Yingtao Gu Ducklings Infected group and control group two groups, on the 7 day old Ducklings Infected group of ducklings subcutaneous injection of duck reovirus (strain HP20090318) 0.2m L/ plume, the control group was injected with physiological saline respectively. After inoculation in 1D, 3D, 5D, 7d, 14d were killed, killed 3 birds each. The eye view pathological observation, 1D and 3D after inoculation of ducklings spleen surface distribution of the size of the red spots, the distribution of 5D and 7d surface around the gray duckling spleen lesions with red spots; the bursa and thymus had no obvious eye lesions. The emergence of a large number of red blood cells, tissue lesions visible after inoculation of 1D and 3D in splenic tissue of red pulp, the white pulp is unclear, after inoculation of 5D and 7d in tissue necrosis and granuloma; group bursal lymph follicle number reduce infection, empty no bubble; thymus See the obvious lesions. Using the immunohistochemical staining results showed that duck reovirus in.RT-PCR spleen macrophages and focal necrosis in detection of duck reovirus S2 gene found in 1D after inoculation, 3D, 5D, 7d and 14d had DRV infection group of ducklings spleen. The results showed that duck reovirus the success of.2.TLR3 infection and replication model, IRF3, RIG-I, duck TRIF, IFN beta signaling molecule m RNA expression (1) TLR3 antiviral signaling pathway, the expression level of TLR3 in 1D after inoculation, and the direct activation of TRIF in the body of 3D, 5D, 7d and 14d expression was no significant difference in the spleen, thymus. Three and bursal tissues, the expression level of each signal molecule in the bursa is higher than that of spleen and thymus.IRF3 is a common intermediate molecular TLR3 signaling pathway and RIG-I signaling pathway, the virus in spleen and bursa in the relative expression level of 3D and 1D increased significantly after inoculation compared, After inoculation of 5D, 7d and 14d relative expression level decreased gradually after inoculation with 1D but the difference was not significant, and the relative expression level in different infection age bursa was higher than the corresponding age of spleen; IRF3 inoculation in thymus tissue after 1D was up-regulated and the other day up not obvious. (2) the expression level of RIG-I RIG-I in antiviral signaling than TLR3 signaling molecules; the expression level in the bursa is higher than that of spleen and thymus in 3D after inoculation, the expression level of 5D is highest. (3) IFN two beta pathway activator mediated and finally, antiviral effect, its relative expression in the spleen, thymus and bursa of three organizations in the water were increased after inoculation, 3D and 5D relative expression level increased. Also in the bursa of the relative expression level is higher than that of spleen and thymus tissue of two. The results of this study show that: the success of copy duck reovirus Model of isolated virus strain HP20090318 in Ducklings Infected animal; duck reovirus in ducklings were identified mainly by the RIG-I, as the main antiviral response pathway; in the spleen, thymus and bursa of three tissues, the expression level of each signal molecule in the bursa was higher than that of spleen and thymus gland, bursa of speculation play the main function in anti duck reovirus role.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.32

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