GPR97对EAE小鼠发病的影响及其对DC细胞的免疫调节作用和机制研究

发布时间：2018-05-13 06:33

本文选题：GPR97 + 基因敲除　；参考：《华东师范大学》2014年硕士论文

【摘要】：G蛋白偶联受体(G protein-coupled receptors, GPCRs),又称为七次跨膜受体,是目前已知的细胞表面第一大类受体家族,在将细胞外信号传入到细胞内起着重要的作用。GPCR在人体中数量极多,其基因数量约占基因组的2%,介导多种生理学反应,发生突变会导致多种疾病,是现在病理学和药物学集中研究的焦点。GPR97属于G蛋白偶联受体2类家族,包含549个氨基酸残基和一段较长的胞外N段,属于孤儿粘附性G蛋白偶联受体。目前其功能尚不明确。由于已知的粘附性GPCRs都与免疫功能存在较大关系,因此GPR97也可能具有免疫调节功能。多发性硬化症(Multiple sclerosis, MS)是一种中枢神经系统的自身免疫性疾病,是导致青壮年人群非创伤性残疾的首要疾病。其发病原因与多种因素有关,包括遗传、环境和自身免疫等,因此,尽管研究较多,但尚未找到有效的治愈手段。EAE (Experimental Autoimmune Encephalomyelitis)是常用的研究MS的动物模型,在发病机理和临床病理方面与MS有很多相似之处。通常采用MOG多肽免疫小鼠的方法制备EAE模型,已经被广泛应用于MS的发病机理及寻找有效治疗方法的研究之中。树突状细胞(Dendritic cell)是人体内最重要的抗原递呈细胞,是连接固有免疫和获得性免疫的重要桥梁。未成熟的DC主要行使抗原吞噬和加工功能,成熟的DC高表达MHC分子和共刺激分子如CD40、CD86等,并能通过分泌不同的细胞因子诱导初始Th细胞向不同的细胞方向进行分化。大量的研究表明,DC细胞可能与EAE的发病有密切的关系。GPR97基因敲除小鼠是研究GPR97基因功能的有力工具。我们检测了GPR97基因敲除小鼠脾细胞中的免疫细胞及其亚群。结果显示,GPR97基因敲除小鼠脾细胞中的T细胞数量略低于野生型小鼠,而NK细胞、CD4+T细胞及Thl和Th17型细胞数量均略高于野生型小鼠。树突状细胞的检测结果表明,GPR97基因敲除小鼠脾脏中的树突状细胞数量显著高于野生型小鼠,而且具有更高的表面分子的表达。为了研究GPR97在EAE发病进程中的作用,我们利用GPR97基因敲除的小鼠为研究对象,采用MOG多肽免疫法建立了EAE模型,通过分析发病小鼠的临床评分、脊髓组织的病理切片及相关免疫学指标的检测来判定GPR97基因敲除对EAE小鼠发病的影响作用。结果显示,与野生型小鼠的EAE模型相比,GPR97基因敲除小鼠的EAE模型临床评分更高,中枢神经系统中炎症细胞浸润现象更加严重,脾脏中Th1和Th17细胞数量更多,炎症因子TNF-α、IL-17A和IL-6表达水平更高,MOG特异性T细胞数量更多,初步证明了GPR97基因敲除的小鼠EAE的发病程度更加严重。为了进一步探讨GPR97影响EAE发病可能的机制,我们以DC细胞为靶细胞,检测了GPR97基因敲除对DC细胞的细胞因子分泌谱、表面分子的表达、诱导Th的分化、抗原递呈等生物学功能的影响。RT-PCR和ELISA检测结果显示,GPR97基因敲除的DC能分泌更多IL-6、TGF-β和IL-12,而IL-10的分泌量减少；GPR97基因敲除的DC细胞主要诱导Th细胞向Th1和Th17细胞分化；GPR97基因敲除的DC细胞表面分子CD40和CD86的表达明显增多；GPR97基因敲除的DC细胞具有更强的刺激MOG特异性T细胞增殖的能力。这些结果表明GPR97基因敲除增强了DC细胞的生物学功能。因此,本研究结果表明GPR97基因与EAE小鼠的发病有关,其可能的机制是GPR97基因敲除增强了DC的免疫功能。该研究为探讨以GPR97为靶点进行MS的治疗奠定了一定的理论和实验基础。
[Abstract]:The G protein coupling receptor (G protein-coupled receptors, GPCRs), also known as the seven transmembrane receptor, is the first known family of receptors on the surface of the cell. It plays an important role in the introduction of extracellular signals into cells, and the number of.GPCR is very large in the human body. The number of genes is about 2% of the genome, which mediates a variety of physiological responses. Mutation can lead to a variety of diseases, and is the focus of the current centralized study of pathology and pharmacology..GPR97 belongs to the 2 family of G protein coupled receptors, including 549 amino acid residues and a long extracellular N segment, belonging to an orphan adherent G protein coupled receptor. GPR97 may also have immune regulation. Multiple sclerosis (MS) is an autoimmune disease of the central nervous system. It is the primary disease that causes non traumatic disability in young and young people. The cause of the disease is related to a variety of factors, including heredity, environment and autoimmunity. Many studies have been made, but the effective cure method.EAE (Experimental Autoimmune Encephalomyelitis) is a common animal model for studying MS. It has many similarities with MS in pathogenesis and Clinicopathology. It is usually used to prepare EAE model by using MOG peptide to immunize mice. It has been widely used in the pathogenesis of MS and it has been widely used in the pathogenesis of MS. In the study of effective treatment methods, Dendritic cell is the most important antigen presenting cell in human body. It is an important bridge to connect inherent immunity and acquired immunity. Immature DC mainly exercises antigen phagocytosis and processing function, mature DC high expression MHC molecule and co stimulator such as CD40, CD86 and so on, and can pass through Different cytokines are secreted to induce initial Th cells to differentiate into different cell directions. A large number of studies have shown that DC cells may have a close relationship with the pathogenesis of EAE..GPR97 knockout mice are a powerful tool to study the function of the GPR97 gene. We detected the immune cells and subgroups in the splenocytes of the GPR97 gene knockout mice. The results showed that the number of T cells in the splenocytes of GPR97 knockout mice was slightly lower than that of the wild type mice, while the number of NK cells, CD4+T cells and Thl and Th17 cells were slightly higher than those of the wild type mice. The detection results of dendritic cells showed that the number of dendritic cells in the spleen of the GPR97 gene knockout mice was significantly higher than that of the wild type mice, and the number of dendritic cells in the spleen was significantly higher than that of the wild type mice. There is a higher expression of surface molecules. In order to study the role of GPR97 in the pathogenesis of EAE, we use the GPR97 gene knockout mice as the research object and use the MOG peptide immunization to establish the EAE model. By analyzing the clinical score of the infected mice, the pathological section of the spinal cord and the detection of the related immunological indexes to determine the GPR97 gene. The effect of knockout on the pathogenesis of EAE mice showed that compared with the EAE model in the wild type mice, the EAE model of GPR97 knockout mice was higher in clinical score, the inflammatory cell infiltration in the central nervous system was more serious, the number of Th1 and Th17 cells in the spleen was more, the expression level of inflammatory factors TNF- a, IL-17A and IL-6 was higher, MOG special. The number of heterosexual T cells is more, and it is preliminarily proved that the incidence of EAE in mice with GPR97 knockout is more serious. In order to further explore the possible mechanism of GPR97 affecting the pathogenesis of EAE, we use DC cells as the target cells to detect the cytokine secreting spectrum of DC cells, the expression of the surface molecules, the differentiation of Th and the antigen presentation of the GPR97 gene knockout. The effects of biological functions such as.RT-PCR and ELISA showed that the GPR97 knockout DC secreted more IL-6, TGF- beta and IL-12, and the secretion of IL-10 decreased; the GPR97 knockout DC cells mainly induced the Th cells to differentiate into Th1 and cells; The PR97 gene knockout DC cells have a stronger ability to stimulate the proliferation of MOG specific T cells. These results suggest that the GPR97 gene knockout enhances the biological function of DC cells. Therefore, the results of this study suggest that the GPR97 gene is associated with the pathogenesis of EAE mice. The possible mechanism of this study is that GPR97 based knockout enhances the immune function of DC. Objective to explore a theoretical and experimental basis for the treatment of MS with GPR97 as the target.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R744.51

【共引文献】