树突状细胞抗原交叉递呈相关SNARE分子靶点的筛选

发布时间：2018-09-12 10:19

【摘要】：背景:抗原的交叉递呈是指外源性抗原进入MHC-Ⅰ类分子处理途径的现象,能够特异性启动细胞毒T细胞应答,构成防御病毒、肿瘤和胞外病原微生物的重要一环,而且还参与维持机体免疫耐受的形成,与多种自身免疫性疾病的发病机制有关。树突状细胞抗原交叉递呈被认为是机体进行外源性抗原交叉递呈的主要途径。外源性抗原的交叉递呈是一系列胞内囊泡融合、转运的过程,SNARE(Soluble-N-ethylmaleimide-sensitive-factor Attachment-protein Receptor)蛋白家族作为一类与囊泡识别、锚定、融合相关的功能蛋白,广泛参与了这一过程。目的:目前对SNARE蛋白的参与交叉递呈的具体机制还知之甚少。本课题率先在这一领域进行探索,为理解树突状细胞抗原交叉递呈的作用机制提供了新的思路。同时,鉴定出的SNARE蛋白将为基于树突状细胞交叉递呈的免疫治疗提供新的靶点。方法:本课题利用siRNA技术对影响DC交叉递呈的SNARE分子进行大规模筛选。首先,利用特异性抗原蛋白OVA刺激APC和T细胞杂交瘤,联合ELISA方法建立起了一套稳定的交叉递呈能力检测体系。然后,收集已知的SNARE分子家族成员,并利用它们的cDNA进行siRNA的设计。接着,利用慢病毒感染的方法将siRNA转入真核的DC2.4细胞中。最后利用稳定表达siRNA的DC2.4细胞和交叉递呈能力检测体系对潜在的能够影响交叉递呈的SNARE分子进行筛选。结果:本课题最终筛选到23个与DC细胞抗原交叉递呈相关的SNARE分子。这些分子主要分属于2个不同的SNARE亚家族,其中Syntaxin3、Syntaxin17和Syntaxin18对交叉递呈的影响较大。结论:作为一类与囊泡识别、锚定、融合相关的功能蛋白,SNARE分子确实参与并影响了DC的外源性抗原交叉递呈。这一结果为免疫调节、病毒防治和肿瘤治疗等方面提供了新的理论支持和作用靶点。
[Abstract]:Background: the cross-presentation of antigens refers to the phenomenon of exogenous antigens entering the MHC- class I molecular processing pathway, which can specifically initiate cytotoxic T cell response and form an important part of the defense against viruses, tumors and extracellular pathogenic microorganisms. It is also involved in the formation of immune tolerance, which is related to the pathogenesis of many autoimmune diseases. Dendritic cell antigen cross-presentation is considered to be the main way to carry out cross-presentation of exogenous antigen. The cross presentation of exogenous antigen is a series of intracellular vesicle fusion, and the translocation process of SNARE (Soluble-N-ethylmaleimide-sensitive-factor Attachment-protein Receptor) protein family as a class of vesicle recognition, anchoring, fusion related functional proteins, widely involved in this process. Objective: little is known about the mechanism of SNARE protein involved in cross-presentation. This topic is the first to explore in this field, which provides a new idea for understanding the mechanism of dendritic cell antigen cross-presentation. At the same time, the identified SNARE protein will provide a new target for immunotherapy based on dendritic cell cross-presentation. Methods: siRNA technique was used to screen large-scale SNARE molecules affecting DC cross-presentation. First, the specific antigen protein OVA was used to stimulate APC and T cell hybridoma, and a stable cross-presentation capacity detection system was established with ELISA method. Then, the known members of the SNARE molecular family are collected and their cDNA is used to design the siRNA. Then, siRNA was transferred into eukaryotic DC2.4 cells by lentivirus infection. Finally, DC2.4 cells expressing siRNA stably and cross-presenting ability detection system were used to screen potential SNARE molecules that could affect cross-presentation. Results: 23 SNARE molecules associated with DC cell antigen cross-presentation were screened. These molecules mainly belong to two different SNARE subfamilies, among which Syntaxin3,Syntaxin17 and Syntaxin18 have great influence on cross-presentation. Conclusion: as a kind of functional protein associated with vesicle recognition, anchoring and fusion, SNARE does participate in and influence the cross-presentation of exogenous antigens in DC. These results provide new theoretical support and target for immunomodulation, virus prevention and cancer therapy.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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