通过免疫Ro60多肽的方法建立一种新的干燥综合征小鼠模型

发布时间：2018-03-19 17:29

本文选题：干燥综合征　切入点：Ro60　出处：《厦门大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：干燥综合征是一种自身免疫病,它影响外分泌腺而导致眼干和口干等症状。这个疾病的致病自身抗原还不清楚,所以目前还缺乏一个好的通过主动免疫抗原而建立的动物模型。干燥综合征病人的一个主要特点是血清中存在抗SSA/Ro60自身抗体,且该自身抗体是诊断干燥综合征标准之一。本研究旨在通过主动免疫Ro60抗原表位多肽,建立类似人类干燥综合征的小鼠模型并探索Ro60抗原在干燥综合征发病机制中的作用。方法：利用Titermax和CFA当佐剂,分别与Ro60_316-335,Ro60_480-494抗原表位短肽混合后脚掌或皮下免疫BALB/c小鼠,对照组用PBS与各佐剂混合后免疫。利用ELISA的方法,我们检测血清抗Ro60多肽IgG抗体以及它的亚型还有血清中的细胞因子的表达情况。在免疫前,免疫后45天和免疫后90/120天,分别检测各组小鼠唾液和泪液分泌量以及体重变化情况。实验在免疫后90/120天结束,从小鼠体内收获的泪腺,唾液腺和其他组织,用来做组织化学和免疫学的评估。用免疫荧光的方法来鉴定组织中的炎症细胞浸润情况。同时,通过免疫荧光的方法来检测组织中自身抗体的沉积情况。最后,.通过转移免疫小鼠的IgG到正常小鼠来检验被免疫小鼠体内抗体的致病性。结果：Ro60_316-335短肽与Titermax佐剂免疫的BALB/c小鼠,在免疫后90天出现了类似干燥综合征的症状。小鼠的泪液分泌量减少,泪腺组织上出现明显的黑色斑点,病理学显示泪腺中腺泡形态改变,腺泡萎缩并出现多处空腔。血清中产生了抗Ro60_316-335短肽的自身抗体,泪腺组织中也可以观察到有自身抗体IgG沉淀。同时,细胞因子水平发生明显变化,其中促炎症因子IFN-γ和IL-17的水平有明显的提高。同时,在泪腺组织中可以观察到T细胞,B细胞,巨噬细胞和中心粒细胞浸润。与免疫Ro60_316-335短肽的小鼠不同的是：免疫Ro60_480-494多肽免疫小鼠和对照组小鼠并没出现以上的症状。最后,如果把佐剂改变为CFA佐剂后,Ro60_316-335多肽免疫的小鼠仅产生抗Ro60_316-335多肽的自身抗体,但并无其它病理和功能改变。而转移免疫小鼠的IgG到正常小鼠并不能导致小鼠外分泌功能降低。结论：通过Ro60_316-335短肽与Titermax佐剂免疫,我们成功地建立了一个新的干燥综合征的小鼠模型。该模型为进一步研究干燥综合征的发病机制及诊断治疗方法奠定了基础。同时,这个模型的成功建立也证明了针对Ro60的自身免疫反应在干燥综合征的发病过程中起到了一定的作用。而这个作用不仅和Ro60蛋白上特定的T细胞多肽表位相关,而且和辅助免疫的佐剂相关。
[Abstract]:Objective: Sjogren's syndrome is an autoimmune disease that affects exocrine glands and causes symptoms such as dry eyes and dry mouth. So there is still a lack of a good animal model established by active immune antigens. One of the main characteristics of Sjogren's syndrome patients is the presence of autoantibodies to SSA/Ro60 in the serum. The aim of this study was to establish a mouse model similar to human Sjogren's syndrome and to explore the role of Ro60 antigen in the pathogenesis of Sjogren's syndrome. Methods: Titermax and CFA were used as adjuvants to immunize BALB/c mice on foot or subcutaneously respectively after mixing with Ro60316-335RoS60 480-494 epitope short peptides, while the control group was immunized with PBS and adjuvants. ELISA method was used. We detected the expression of serum anti Ro60 polypeptide IgG antibody, its subtype and cytokines in the serum. Before immunization, 45 days after immunization and 90/120 days after immunization, The changes of saliva and tear secretion and body weight of each group were measured respectively. After 90/120 days of immunization, lacrimal glands, salivary glands and other tissues were harvested from the mice. Used for histochemical and immunological assessments. Immunofluorescence was used to identify the infiltration of inflammatory cells in the tissue. At the same time, Autoantibodies in tissues were detected by immunofluorescence method. Finally, the pathogenicity of antibodies in immunized mice was examined by transferring IgG from immunized mice to normal mice. Results in BALB/c mice immunized with Titermax adjuvant, the symptoms of Sjogren's syndrome appeared 90 days after immunization. The amount of tear secretion in mice decreased, and there were obvious black spots in lacrimal gland tissue. Pathological changes in the lacrimal gland showed acinar morphology, acinar atrophy and multiple cavities. Autoantibodies against Ro60_316-335 short peptides were produced in serum, and autoantibodies IgG precipitated in lacrimal gland tissues. The levels of cytokines, IFN- 纬 and IL-17, were significantly increased. At the same time, T cell B cells were observed in lacrimal gland. Macrophages and central granulocytes infiltrate. Unlike mice immunized with Ro60_316-335 peptides, the mice immunized with Ro60_480-494 peptides did not show these symptoms. If the adjuvant was changed to CFA adjuvant, the mice immunized with Ro60316-335 polypeptide only produced autoantibodies against Ro60_316-335 polypeptide, but there were no other pathological and functional changes. However, the mice transferred to normal mice could not reduce the exocrine function of mice. Conclusion: we successfully established a new Sjogren syndrome mouse model by immunizing with Ro60_316-335 short peptide and Titermax adjuvant, which laid a foundation for further study on pathogenesis and diagnosis and treatment of Sjogren syndrome. The successful establishment of this model also demonstrated that the autoimmune response to Ro60 may play a role in the pathogenesis of Sjogren's syndrome, which is related not only to the specific T cell polypeptide epitopes on the Ro60 protein. And associated with adjuvants of adjuvant immunity.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R-332;R593.2

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