本文選題：鴨呼腸孤病毒　切入點：TLR3　出處：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：鴨呼腸孤病毒(Duck reovirus,DRV)感染雛鴨后,主要侵害雛鴨免疫系統(tǒng),如脾、法氏囊、胸腺,引起雛鴨免疫器官損傷,從而繼發(fā)其它細菌感染。天然免疫系統(tǒng)中有很多模式識別受體可以識別病毒核酸,從而激活機體的抗病毒反應(yīng)。TLR3和RIG-I可以特異性的識別侵入機體的雙鏈RNA病毒,激活下游的信號分子,最終誘導(dǎo)IFNβ的釋放,引起抗病毒免疫反應(yīng)。目前,對鴨呼腸孤病毒誘導(dǎo)抗病毒反應(yīng)的信號通路還不明確,本研究通過病毒感染動物模型的復(fù)制,研究鴨呼腸孤病毒感染雛鴨后兩條抗病毒反應(yīng)信號通路中相關(guān)分子的m RNA表達變化,初步闡明病毒引起的主要抗病毒天然免疫信號途徑。主要研究內(nèi)容如下:1.鴨呼腸孤病毒感染雛鴨模型的復(fù)制將30羽櫻桃谷雛鴨隨機均分為感染組和對照組兩組,于雛鴨7日齡對感染組雛鴨頸部皮下注射鴨呼腸孤病毒(HP20090318株)0.2m L/羽,對照組注射等量的生理鹽水。分別于接毒后1d、3d、5d、7d、14d進行剖殺,每組剖殺3羽。眼觀病理學(xué)觀察,接毒后1d和3d雛鴨脾表面分布有大小不一的紅色斑點,5d和7d雛鴨脾表面分布有灰白色病灶病灶周圍有紅色斑點;法氏囊與胸腺未見明顯眼觀病變。組織病變可見接毒后1d和3d脾組織內(nèi)出現(xiàn)大量紅細胞,紅髓、白髓界限不清,接毒后5d和7d在組織內(nèi)出現(xiàn)壞死灶及肉芽腫;感染組法氏囊淋巴濾泡數(shù)目減少,出現(xiàn)空泡;胸腺組織內(nèi)未見明顯病變。用免疫組化染色結(jié)果顯示鴨呼腸孤病毒存在于脾巨噬細胞以及壞死灶中。RT-PCR檢測鴨呼腸孤病毒S2基因,發(fā)現(xiàn)在接毒后1d、3d、5d、7d以及14d感染組雛鴨脾均有DRV存在。上述結(jié)果表明鴨呼腸孤病毒感染雛鴨模型復(fù)制成功。2.TLR3、TRIF、IRF3、RIG-I、IFNβ信號分子m RNA表達變化檢測(1)TLR3抗病毒信號通路中,TLR3與其直接激活的TRIF在機體內(nèi)表達水平在接毒后1d、3d、5d、7d以及14d表達差異不明顯,在脾、胸腺與法氏囊三個組織中,法氏囊內(nèi)各信號分子的表達水平高于脾和胸腺。IRF3是TLR3信號通路與RIG-I信號通路中共同的中間分子,其在脾與法氏囊中接毒后3d的相對表達水平與接毒后1d相比上升明顯,接毒后5d、7d以及14d相對表達水平逐漸下降但與接毒后1d相比差異不明顯,并且在不同感染日齡法氏囊內(nèi)的相對表達水平均高于相應(yīng)日齡的脾;IRF3在胸腺組織中接毒后1d表達明顯上調(diào),其他日齡上調(diào)不明顯。(2)RIG-I抗病毒信號通路中RIG-I的表達水平要高于TLR3信號分子;其中在法氏囊內(nèi)的表達水平高于脾和胸腺,在接毒后3d、5d表達水平最高。(3)IFNβ是兩條信號通路的最終激活因子,并介導(dǎo)抗病毒效應(yīng),其在脾、胸腺與法氏囊三個組織中的相對表達水均呈現(xiàn)上調(diào)趨勢,接毒后3d和5d相對表達水平上調(diào)。同樣在法氏囊內(nèi)的相對表達水平要高于脾和胸腺兩組織。本研究結(jié)果表明:成功復(fù)制了鴨呼腸孤病毒HP20090318株感染雛鴨動物模型;鴨呼腸孤病毒進入雛鴨體內(nèi)主要由RIG-I進行識別,為主要的抗病毒反應(yīng)通路;在脾、法氏囊和胸腺三個組織中,法氏囊內(nèi)各信號分子的表達水平要高于脾和胸腺,推測法氏囊在抗鴨呼腸孤病毒的作用中發(fā)揮著主要功能。
[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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.32

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