發(fā)布時(shí)間：2018-04-14 03:17

  本文選題：IBDV + chTLR3信號(hào)轉(zhuǎn)導(dǎo)��； 參考：《東北農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：為了探討chTLR3信號(hào)轉(zhuǎn)導(dǎo)通路在傳染性法氏囊病病毒(IBDV)致雛雞法氏囊免疫損傷機(jī)制中的作用,本試驗(yàn)將75只SPF雛雞隨機(jī)分為3組,每組25只,分別為感染組、疫苗組和對(duì)照組。嚴(yán)格隔離飼養(yǎng)。感染組雛雞經(jīng)點(diǎn)眼滴鼻途徑,感染IBDV標(biāo)準(zhǔn)毒株,0.6ml/只。疫苗組經(jīng)相同途徑給予雛雞IBDV活疫苗,2羽份/只。對(duì)照組經(jīng)相同途徑給予PBS,0.6ml/只。于感染后第1、4、7、21及35天每組隨機(jī)抽取5只雛雞心臟采血處死,快速采取法氏囊于-80℃保存。采用實(shí)時(shí)熒光定量PCR技術(shù)對(duì)法氏囊中的chTLR3、chTLR3信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān)信號(hào)分子(ch MyD88、ch TRIF和ch NF-κB)及細(xì)胞因子(chTNF-α、chIL-1β、chIL-6、chIFN-β)的mRNA表達(dá)變化進(jìn)行檢測(cè);采用間接免疫熒光技術(shù)對(duì)法氏囊中的chTLR3蛋白表達(dá)以及T、B淋巴細(xì)胞、巨噬細(xì)胞數(shù)量變化進(jìn)行檢測(cè)。結(jié)果發(fā)現(xiàn),感染組與疫苗組雛雞法氏囊中chTLR3 mRNA的相對(duì)表達(dá)量及其蛋白表達(dá)量在感染IBDV或接種IBDV活疫苗后均呈現(xiàn)先上升后降低的趨勢(shì),并于第4天達(dá)到峰值(P0.01或P0.05)。表明ch TLR3參與IBDV感染雛雞的過程。感染組雛雞法氏囊中的chTLR3信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān)信號(hào)分子的相對(duì)表達(dá)量在感染初期均顯著高于對(duì)照組(P0.01或P0.05),并皆于第4天達(dá)到峰值,之后逐漸下降。其中,ch MyD88與ch TRIF相比上調(diào)的幅度小、時(shí)間短,表明chTLR3主要接頭蛋白分子為ch TRIF。感染組及疫苗組雛雞法氏囊中細(xì)胞因子的表達(dá)趨勢(shì)與chTLR3信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān)信號(hào)分子基本一致,即呈現(xiàn)先上調(diào)后下降的規(guī)律。適應(yīng)性免疫相關(guān)指標(biāo)結(jié)果顯示,感染組雛雞感染IBDV后第1-7d,法氏囊中T淋巴細(xì)胞數(shù)量逐漸增加且極顯著高于對(duì)照組(P0.01),但于第21天顯著低于對(duì)照組(P0.05),第35天雖有所上升但仍顯著低于對(duì)照組(P0.05);疫苗組雛雞接種IBDV疫苗后法氏囊中T淋巴細(xì)胞數(shù)量于接種后第1天開始上升,第4天達(dá)到峰值(P0.05),之后逐漸下降,于第21天恢復(fù)正常(P0.05)。結(jié)果顯示,感染初期細(xì)胞因子相對(duì)表達(dá)量與T淋巴細(xì)胞數(shù)量均呈上升趨勢(shì)。感染組雛雞法氏囊中B淋巴細(xì)胞數(shù)量在感染IBDV后第1天便極顯著低于對(duì)照組(P0.01)并逐漸降低,第21天雖有所升高但仍然極顯著低于對(duì)照組(P0.01),之后逐漸恢復(fù);疫苗組雛雞法氏囊B淋巴細(xì)胞數(shù)量在接種后第1-7d均顯著低于對(duì)照組(P0.05),但整體呈現(xiàn)逐漸升高狀態(tài)。結(jié)果顯示,在感染后期細(xì)胞因子(IFN-β除外)相對(duì)表達(dá)量與B淋巴細(xì)胞數(shù)量均呈上升趨勢(shì)。上述結(jié)果表明,chTLR3可通過下游細(xì)胞因子在感染早期參與誘導(dǎo)細(xì)胞免疫的發(fā)生,在感染晚期參與誘導(dǎo)體液免疫的發(fā)生。另外,感染組雛雞巨噬細(xì)胞數(shù)量在感染后第1-7d顯著高于對(duì)照組(P0.01或P0.05),第21天顯著低于對(duì)照組(P0.05),第35天恢復(fù)。疫苗組雛雞巨噬細(xì)胞數(shù)量呈先升高后降低趨勢(shì),于接種后第7天恢復(fù)。結(jié)果顯示,chTLR3相對(duì)表達(dá)量與巨噬細(xì)胞、T淋巴細(xì)胞數(shù)量在感染前期均升高,提示在感染早期chTLR3可通過誘導(dǎo)巨噬細(xì)胞的激活而間接激活細(xì)胞免疫。本試驗(yàn)從基因及蛋白水平證明,chTLR3在IBDV感染雛雞及雛雞抗感染過程中扮演重要角色,chTLR3信號(hào)通路通過激活下游細(xì)胞因子及巨噬細(xì)胞調(diào)節(jié)獲得性免疫,進(jìn)一步證明了chTLR3是連接天然免疫與獲得性免疫的橋梁,為研究模式識(shí)別受體在抗病毒反應(yīng)中的作用提供了科學(xué)的試驗(yàn)依據(jù),為傳染性法氏囊病的防治提供了新的思路。
[Abstract]:In order to investigate the chTLR3 signal transduction pathway in infectious bursal disease virus (IBDV) induced immune injury mechanism of the bursal role in this experiment 75 SPF chickens were randomly divided into 3 groups, 25 rats in each group, respectively. The infection group, the vaccine group and control group. Strictly kept in isolation. The eye nose drop methods group of chickens infected with IBDV infection, virus, 0.6ml/ vaccine group. Only by the same way of giving the chicks IBDV vaccine, 2 samples of each. Control group by the same way to PBS, 0.6ml/. After infection at day 1,4,7,21 and 35 randomly selected in each group and 5 chickens were killed to quickly take heart, bursa in -80 C save. Using the real-time quantitative PCR on Bursa in chTLR3, chTLR3 signal transduction pathway related signal molecules (CH MyD88, CH TRIF and CH NF- K B) and cytokines (chTNF- alpha, chIL-1 beta, chIL-6, chIFN-) was used to detect the expression change of mRNA; the The expression of chTLR3 protein in the bursa and T with immunofluorescence technique B lymphocytes, changes in the number of macrophages were detected. The results showed that the relative expression of infection group and vaccine group bursal chTLR3 mRNA and protein expression were increased and then decreased in IBDV infected or vaccinated with IBDV vaccine after fourth days and reached the peak at (P0.01 or P0.05). The results indicated that CH TLR3 is involved in the process of chickens infected with IBDV infection. ChTLR3 signal transduction pathway related signaling molecules in the bursal group in the relative expression in the early stage of infection were significantly higher than the control group (P0.01 or P0.05), and reached the peak at fourth day, then decreased gradually. Among them, CH MyD88 and CH TRIF compared to the increase of small, short time, indicating that chTLR3 protein molecules called cytokines ch joint TRIF. infection group and the bursal vaccine group the expression of C and trend The hTLR3 signal transduction pathway related signaling molecules are basically the same, which showed increased firstly and then decreased. The rule of adaptive immune related indicators showed that the infection group of chickens infected with IBDV after 1-7d, the number of T lymphocytes in the bursa increased gradually and significantly higher than the control group (P0.01), but in twenty-first days was significantly lower than the control group (P0.05), thirty-fifth days has increased but still significantly lower than the control group (P0.05); the number of T lymphocytes in the vaccine group chicks vaccinated with IBDV vaccine after bursa began first days after inoculation increased, reached the peak at the fourth day (P0.05), then decreased gradually, on the twenty-first day return to normal (P0.05). The results showed that the infection at the beginning of the relative expression quantity of cytokines and T lymphocyte increased. The number of infected B lymphocytes in bursa of Fabricius in first days after infection in IBDV was significantly lower than the control group (P0.01) and decreased gradually, despite twenty-first days Increased but still significantly lower than the control group (P0.01), then gradually recovered; the number of vaccine group bursal B lymphocytes in 1-7d after inoculation were significantly lower than the control group (P0.05), but the overall state has gradually increased. The results showed that in the later stage of infection, cytokines (IFN- beta except) the relative expression quantity and B lymphocytes increased. These results indicate that chTLR3 can be through the downstream cytokines involved in the induction of immune cells in the early stage of infection occurred in the later stage of infection is involved in the induction of humoral immunity. In addition, the number of chickens infected macrophages were significantly higher than those in the control group after infection 1-7d (P0.01 or P0.05), twenty-first days significantly lower than the control group (P0.05), thirty-fifth days of recovery. The number of vaccine group chicken macrophages increased at first and then decreased, on the seventh day after inoculation of recovery. The results showed that the relative expression of chTLR3 and macrophage, T lymphocyte The cell number was increased in early infection, suggesting that in the early stage of infection chTLR3 can be induced by activation of macrophages and indirect activation of immune cells. The gene and protein level, chTLR3 in IBDV chickens and chicken anti infection plays an important role in the process of activation, cytokines and macrophages regulate acquired immunity through chTLR3 signal pathway. Further proved that chTLR3 is a bridge innate and adaptive immunity, provide scientific experimental basis for studying the role of pattern recognition receptors in antiviral response, provides a new idea for the prevention and treatment of infectious bursal disease.

【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S858.31

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