鸡TLR3信号通路在IBDV致雏鸡法氏囊免疫损伤中的作用研究

发布时间：2018-04-14 03:17

本文选题：IBDV + chTLR3信号转导　；参考：《东北农业大学》2016年硕士论文

【摘要】：为了探讨chTLR3信号转导通路在传染性法氏囊病病毒(IBDV)致雏鸡法氏囊免疫损伤机制中的作用,本试验将75只SPF雏鸡随机分为3组,每组25只,分别为感染组、疫苗组和对照组。严格隔离饲养。感染组雏鸡经点眼滴鼻途径,感染IBDV标准毒株,0.6ml/只。疫苗组经相同途径给予雏鸡IBDV活疫苗,2羽份/只。对照组经相同途径给予PBS,0.6ml/只。于感染后第1、4、7、21及35天每组随机抽取5只雏鸡心脏采血处死,快速采取法氏囊于-80℃保存。采用实时荧光定量PCR技术对法氏囊中的chTLR3、chTLR3信号转导通路相关信号分子(ch MyD88、ch TRIF和ch NF-κB)及细胞因子(chTNF-α、chIL-1β、chIL-6、chIFN-β)的mRNA表达变化进行检测;采用间接免疫荧光技术对法氏囊中的chTLR3蛋白表达以及T、B淋巴细胞、巨噬细胞数量变化进行检测。结果发现,感染组与疫苗组雏鸡法氏囊中chTLR3 mRNA的相对表达量及其蛋白表达量在感染IBDV或接种IBDV活疫苗后均呈现先上升后降低的趋势,并于第4天达到峰值(P0.01或P0.05)。表明ch TLR3参与IBDV感染雏鸡的过程。感染组雏鸡法氏囊中的chTLR3信号转导通路相关信号分子的相对表达量在感染初期均显著高于对照组(P0.01或P0.05),并皆于第4天达到峰值,之后逐渐下降。其中,ch MyD88与ch TRIF相比上调的幅度小、时间短,表明chTLR3主要接头蛋白分子为ch TRIF。感染组及疫苗组雏鸡法氏囊中细胞因子的表达趋势与chTLR3信号转导通路相关信号分子基本一致,即呈现先上调后下降的规律。适应性免疫相关指标结果显示,感染组雏鸡感染IBDV后第1-7d,法氏囊中T淋巴细胞数量逐渐增加且极显著高于对照组(P0.01),但于第21天显著低于对照组(P0.05),第35天虽有所上升但仍显著低于对照组(P0.05);疫苗组雏鸡接种IBDV疫苗后法氏囊中T淋巴细胞数量于接种后第1天开始上升,第4天达到峰值(P0.05),之后逐渐下降,于第21天恢复正常(P0.05)。结果显示,感染初期细胞因子相对表达量与T淋巴细胞数量均呈上升趋势。感染组雏鸡法氏囊中B淋巴细胞数量在感染IBDV后第1天便极显著低于对照组(P0.01)并逐渐降低,第21天虽有所升高但仍然极显著低于对照组(P0.01),之后逐渐恢复;疫苗组雏鸡法氏囊B淋巴细胞数量在接种后第1-7d均显著低于对照组(P0.05),但整体呈现逐渐升高状态。结果显示,在感染后期细胞因子(IFN-β除外)相对表达量与B淋巴细胞数量均呈上升趋势。上述结果表明,chTLR3可通过下游细胞因子在感染早期参与诱导细胞免疫的发生,在感染晚期参与诱导体液免疫的发生。另外,感染组雏鸡巨噬细胞数量在感染后第1-7d显著高于对照组(P0.01或P0.05),第21天显著低于对照组(P0.05),第35天恢复。疫苗组雏鸡巨噬细胞数量呈先升高后降低趋势,于接种后第7天恢复。结果显示,chTLR3相对表达量与巨噬细胞、T淋巴细胞数量在感染前期均升高,提示在感染早期chTLR3可通过诱导巨噬细胞的激活而间接激活细胞免疫。本试验从基因及蛋白水平证明,chTLR3在IBDV感染雏鸡及雏鸡抗感染过程中扮演重要角色,chTLR3信号通路通过激活下游细胞因子及巨噬细胞调节获得性免疫,进一步证明了chTLR3是连接天然免疫与获得性免疫的桥梁,为研究模式识别受体在抗病毒反应中的作用提供了科学的试验依据,为传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.

【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.31

【相似文献】