【摘要】：羊接觸傳染性膿皰皮炎,或稱羊口瘡(Contagiouts Ecthyma,CE or ORF),是目前世界上養(yǎng)羊生產(chǎn)中的常見疫病之一,在我國主要養(yǎng)殖地區(qū)吉林、寧夏、云南、河南、湖北、新疆和陜西等均有流行報(bào)道,山羊比綿羊更易感染,山羊養(yǎng)殖場(chǎng)的發(fā)病率通常為10%~100%。雖然該病本身很少導(dǎo)致成年山羊死亡,但是對(duì)新生羔羊危害嚴(yán)重,感染口唇部引起的嚴(yán)重潰爛影響羔羊采食,最終可導(dǎo)致死亡;此外,該病還危害成年羊的生產(chǎn)性能,引起乳房炎等其他疾病的發(fā)生。羊口瘡的臨癥特點(diǎn)為口唇部發(fā)生丘疹、水皰、膿皰、皮膚破潰最終形成結(jié)痂,為典型炎癥反應(yīng),該炎癥過程中有哪些相關(guān)的細(xì)胞和細(xì)胞因子參與,還未見報(bào)道。本研究利用病理組織切片技術(shù)、淋巴細(xì)胞增殖、real-time PCR及原核表達(dá)等方法,采集臨床初次感染羔羊不同發(fā)病時(shí)期口唇部損傷組織,研究羊口瘡病程中上皮組織病變特點(diǎn);利用羊口瘡強(qiáng)、弱毒株分別刺激健康山羊外周血單個(gè)核淋巴細(xì)胞(Peripheral blood mononuclear cells,PBMCs),檢測(cè)PBMCs增殖情況以及細(xì)胞因子mRNA水平;同時(shí),檢測(cè)羊口瘡感染羊病程各時(shí)期PBMCs產(chǎn)生細(xì)胞因子mRNA水平;表達(dá)山羊IL-1β和IL-6蛋白。研究結(jié)果如下:1.羊口瘡引起的皮膚損傷其主要組織學(xué)變化為上皮細(xì)胞空泡樣變性以及基層下結(jié)締組織間炎性細(xì)胞浸潤(rùn)。隨著病程的發(fā)展,浸潤(rùn)細(xì)胞數(shù)目增多,發(fā)展為膿皰期時(shí)浸潤(rùn)細(xì)胞數(shù)目最多,淋巴細(xì)胞浸潤(rùn)顯著,進(jìn)入康復(fù)期后浸潤(rùn)細(xì)胞數(shù)目逐漸降低。病變組織浸潤(rùn)細(xì)胞主要包括淋巴細(xì)胞、中性粒細(xì)胞、巨噬細(xì)胞等免疫細(xì)胞。病變初期浸潤(rùn)的炎性細(xì)胞主要為中性粒細(xì)胞,水皰期和膿皰期主要為淋巴細(xì)胞浸潤(rùn),康復(fù)期的浸潤(rùn)細(xì)胞主要為淋巴細(xì)胞和巨噬細(xì)胞。2.羊口瘡強(qiáng)、弱毒株均能顯著刺激山羊PBMCs增殖,強(qiáng)毒株作用明顯優(yōu)于弱毒株;PBMCs中IL-17 mRNA水平顯著升高,IL-1β、IL-6和IL-23三種細(xì)胞因子mRNA水平升高顯著,TGF-βmRNA水平則呈明顯下降。羊口瘡病程中PBMCs產(chǎn)生細(xì)胞因子mRNA水平檢測(cè)結(jié)果顯示,水泡期IL-1β、IL-6和IL-23 mRNA水平顯著升高,膿皰期IL-17 mRNA水平顯著升高,同時(shí),IFN-γmRNA水平也有明顯的升高。3.克隆山羊IL-1β和IL-6基因,目的片段大小分別為463 bp和552 bp,原核誘導(dǎo)表達(dá)獲得的IL-1β和IL-6融合蛋白,大小分別為37.4 kDa和40.6 kDa,表達(dá)產(chǎn)物為可溶性蛋白。免疫家兔制備多克隆抗體,瓊脂雙擴(kuò)散測(cè)定血清抗體效價(jià)分別為1:16和1:8。本研究結(jié)果表明:1. 羊口瘡病理過程本質(zhì)為免疫病理反應(yīng),參與此過程的免疫細(xì)胞在早期為中性粒細(xì)胞,后期為淋巴細(xì)胞。2. 介導(dǎo)羊口瘡免疫病理反應(yīng)的主要細(xì)胞因子為IL 17。根據(jù)本研究測(cè)定的其它細(xì)胞因子結(jié)果,推測(cè)羊口瘡反應(yīng)過程中的IL 17來源于Th17細(xì)胞;此外,羊口瘡炎癥反應(yīng)過程也有Th1細(xì)胞亞群參與。3. 克隆表達(dá)山羊IL 1β和IL 6蛋白及抗血清,為本實(shí)驗(yàn)室進(jìn)一步探討羊口瘡免疫病理機(jī)制提供了準(zhǔn)備。
[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- 緯 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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.27

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