羊口疮病程中Th17细胞相关细胞因子的变化

发布时间：2018-10-29 16:41

【摘要】：羊接触传染性脓疱皮炎,或称羊口疮(Contagiouts Ecthyma,CE or ORF),是目前世界上养羊生产中的常见疫病之一,在我国主要养殖地区吉林、宁夏、云南、河南、湖北、新疆和陕西等均有流行报道,山羊比绵羊更易感染,山羊养殖场的发病率通常为10%~100%。虽然该病本身很少导致成年山羊死亡,但是对新生羔羊危害严重,感染口唇部引起的严重溃烂影响羔羊采食,最终可导致死亡;此外,该病还危害成年羊的生产性能,引起乳房炎等其他疾病的发生。羊口疮的临症特点为口唇部发生丘疹、水疱、脓疱、皮肤破溃最终形成结痂,为典型炎症反应,该炎症过程中有哪些相关的细胞和细胞因子参与,还未见报道。本研究利用病理组织切片技术、淋巴细胞增殖、real-time PCR及原核表达等方法,采集临床初次感染羔羊不同发病时期口唇部损伤组织,研究羊口疮病程中上皮组织病变特点;利用羊口疮强、弱毒株分别刺激健康山羊外周血单个核淋巴细胞(Peripheral blood mononuclear cells,PBMCs),检测PBMCs增殖情况以及细胞因子mRNA水平;同时,检测羊口疮感染羊病程各时期PBMCs产生细胞因子mRNA水平;表达山羊IL-1β和IL-6蛋白。研究结果如下:1.羊口疮引起的皮肤损伤其主要组织学变化为上皮细胞空泡样变性以及基层下结缔组织间炎性细胞浸润。随着病程的发展,浸润细胞数目增多,发展为脓疱期时浸润细胞数目最多,淋巴细胞浸润显著,进入康复期后浸润细胞数目逐渐降低。病变组织浸润细胞主要包括淋巴细胞、中性粒细胞、巨噬细胞等免疫细胞。病变初期浸润的炎性细胞主要为中性粒细胞,水疱期和脓疱期主要为淋巴细胞浸润,康复期的浸润细胞主要为淋巴细胞和巨噬细胞。2.羊口疮强、弱毒株均能显著刺激山羊PBMCs增殖,强毒株作用明显优于弱毒株;PBMCs中IL-17 mRNA水平显著升高,IL-1β、IL-6和IL-23三种细胞因子mRNA水平升高显著,TGF-βmRNA水平则呈明显下降。羊口疮病程中PBMCs产生细胞因子mRNA水平检测结果显示,水泡期IL-1β、IL-6和IL-23 mRNA水平显著升高,脓疱期IL-17 mRNA水平显著升高,同时,IFN-γmRNA水平也有明显的升高。3.克隆山羊IL-1β和IL-6基因,目的片段大小分别为463 bp和552 bp,原核诱导表达获得的IL-1β和IL-6融合蛋白,大小分别为37.4 kDa和40.6 kDa,表达产物为可溶性蛋白。免疫家兔制备多克隆抗体,琼脂双扩散测定血清抗体效价分别为1:16和1:8。本研究结果表明:1. 羊口疮病理过程本质为免疫病理反应,参与此过程的免疫细胞在早期为中性粒细胞,后期为淋巴细胞。2. 介导羊口疮免疫病理反应的主要细胞因子为IL 17。根据本研究测定的其它细胞因子结果,推测羊口疮反应过程中的IL 17来源于Th17细胞;此外,羊口疮炎症反应过程也有Th1细胞亚群参与。3. 克隆表达山羊IL 1β和IL 6蛋白及抗血清,为本实验室进一步探讨羊口疮免疫病理机制提供了准备。
[Abstract]:Sheep contact with infectious pustular dermatitis, or Contagiouts Ecthyma,CE or ORF), is one of the most common diseases in sheep production in the world. It is widely used in Jilin, Ningxia, Yunnan, Henan and Hubei provinces in China. Xinjiang and Shaanxi provinces have reported that goats are more susceptible to infection than sheep, and the incidence of goat farms is usually 10% and 100%. Although the disease itself rarely leads to adult goat death, but to the newborn lamb serious harm, infection of mouth and lips caused by serious ulcers affect the feeding of lambs, and eventually lead to death; In addition, the disease also endangers the productive performance of adult sheep, causing mastitis and other diseases. The clinical features of sheep mouth ulcers include papules, blisters, pustules, skin rupture and finally scab, which is a typical inflammatory reaction. What kinds of cells and cytokines are involved in the process of this inflammation have not been reported yet. In this study, histopathological techniques, lymphocyte proliferation, real-time PCR and prokaryotic expression were used to study the characteristics of epithelial lesions in the course of sheep mouth ulcers. The healthy goat peripheral blood mononuclear lymphocytes (Peripheral blood mononuclear cells,PBMCs) were stimulated by the strong and weak strains of sheep mouth sore, and the proliferation of PBMCs and the level of cytokine mRNA were detected. At the same time, the levels of cytokine mRNA and the expression of IL-1 尾 and IL-6 protein were detected in sheep infected with aphtha. The results are as follows: 1. The main histological changes of skin injury caused by sheep mouth sore were vacuolar degeneration of epithelial cells and infiltration of inflammatory cells in subbasal connective tissue. With the development of the course of disease, the number of infiltrating cells increased, and the number of infiltrating cells was the most in pemphigus stage, and the number of infiltrating cells decreased gradually after entering the convalescent stage. Infiltrating cells mainly include lymphocytes, neutrophils, macrophages and other immune cells. The infiltration of inflammatory cells was mainly neutrophilic granulocyte in the early stage of the lesion, lymphocyte infiltration in blister and pemphigus, and lymphocyte and macrophage in convalescent stage. 2. Sheep mouth sore was strong and attenuated strains could significantly stimulate goat PBMCs proliferation. The effect of virulent strain was better than that of attenuated strain. The levels of IL-17 mRNA, IL-1 尾, IL-6 and IL-23 in PBMCs increased significantly, while TGF- 尾 mRNA decreased significantly. The level of cytokine mRNA produced by PBMCs in sheep mouth sore course showed that the levels of IL-1 尾, IL-6 and IL-23 mRNA in vesicular phase were significantly increased, IL-17 mRNA level in pemphigus phase was significantly increased, and IFN- 纬 mRNA level was also significantly increased. 3. Goat IL-1 尾 and IL-6 genes were cloned. The target fragments were 463 bp and 552 bp, prokaryotic expression fusion proteins, respectively. The size of IL-1 尾 and IL-6 fusion proteins were 37.4 kDa and 40.6 kDa, respectively. The polyclonal antibodies were prepared by immunizing rabbits. The titers of serum antibodies determined by Agar double diffusion were 1:16 and 1: 8, respectively. The results show that: 1. The pathological process of sheep mouth ulcer is essentially immune pathological reaction. The immune cells involved in this process are neutrophils at the early stage and lymphocytes at the later stage. The main cytokine that mediates the immune pathological reaction of sheep mouth sore is IL. According to the results of other cytokines measured in this study, we speculated that the IL 17 in sheep aphthous ulcer reaction originated from Th17 cells, in addition, Th1 cell subsets were also involved in the inflammatory reaction of sheep aphtha. 3. The cloning and expression of goat IL 1 尾, IL 6 protein and antiserum provide the preparation for further study of the immune pathological mechanism of sheep aphthous ulcer in our laboratory.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.27

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