Rab5和Rab7与抗原的交叉递呈研究

发布时间：2018-12-18 05:53

【摘要】： 交叉递呈是外来抗原被MHC-Ⅰ类分子限制性递呈的过程,它不同与经典的MHC-Ⅰ类分子递呈途径,在经典的MHC递呈途径中,内源性表达蛋白经蛋白酶体消化后,小部分的降解肽通过TAP进入内质网,在内质网中与新生成的MHC-Ⅰ类分子形成复合物转运至细胞膜,相反MHC-Ⅱ类分子的递呈途径则主要处理进入内吞系统的外来抗原,这样MHC-Ⅰ和MHC-Ⅱ类分子分管着胞内和胞外不同来源的抗原。在体情况下,DC细胞是主要的交叉递呈细胞,它通过在外周获取感染性细胞的内源性抗原,然后迁移到淋巴结,形成的MHC-Ⅰ类分子肽复合物活化抗原特异性的细胞毒性T细胞,在此过程中抗原的来源(细胞内,但是不同的细胞,或者疫苗的接种中的外来抗原)不同于经典的MHC-Ⅰ类分子递呈途径(在抗原递呈细胞内合成的抗原)。因此,在上述抗原递呈中,抗原的降解和肽的运送机制很可能显著不同。近年来,由于认识到交叉递呈在疫苗接种中的重要作用,因此其机制研究引起了广泛关注。胞内膜蛋白运输通常需要含有Rab蛋白的蛋白复合物,其中Rab蛋白是真核生物膜转运事件的关键分子。人类基因组共含有大约70个Rab和Rab样蛋白,它们通常位于不同的膜性结构区域,采用鸟苷酸依赖的开关机制调节膜质运输的四个步骤:囊泡的出芽生成,囊泡的转运,囊泡的栓缚和囊泡与靶膜结构的融合。现有证据显示有几个蛋白位于早期的分拣和回运囊泡(Rab4,Rab5,Rab11,Rab18,Rab22 andRab25),而Rab7和Rab9定位于晚期内吞体,至今在树突状细胞的抗原递呈中,尚无人进行Rab分子的相关研究,因此,本文尝试在树突状细胞中进行此相关研究。本研究希望通过转染Rab5和Rab7突变体,在树突状细胞中高表达Rab5,Rab7和他们的突变体,观察与囊泡转运相关的Rab分子,从而了解抗原的交叉递呈主要发生在吞噬体成熟过程的什么阶段。首先使用Rab5的抑制型突变体Rab5S34N和Rab7的抑制型突变体Rab7T22N观察这两个蛋白对抗原交叉递呈的影响,结果表明Rab5S34N抑制了抗原的交叉递呈,但Rab7T22N对抗原交叉递呈则没有影响。为近一步分析Rab5在此过程中的作用,我们构建了Rab5的活化型突变体RabSQ79L,结果表明其抗原递呈效果与Rab5野生型一致,这说明早期内吞体是抗原交叉递呈发生的主要场所,此研究结果为进一步分析抗原交叉递呈过程中囊泡的转运和调节奠定了基础。
[Abstract]:Cross presentation is a process in which foreign antigens are restricted by MHC- class I molecules. It is different from the classical MHC- class I molecular presentation pathway. In the classical MHC presentation pathway, endogenous expressed proteins are digested by proteasome. A small portion of the degradation peptides enter the endoplasmic reticulum through TAP and form complexes with newly formed MHC- class I molecules in the endoplasmic reticulum. On the contrary, the presentation pathway of MHC- class II molecules mainly deals with the foreign antigens entering the endocytosis system. Thus, MHC- I and MHC- II molecules are responsible for different antigens from both intracellular and extracellular sources. In vivo, DC cells are the primary cross-presenting cells, which obtain the endogenous antigen of the infected cells at the peripheral level and migrate to the lymph nodes. The formation of MHC- class I peptide complexes activates antigen-specific cytotoxic T cells, during which antigen sources (within cells, but different cells), Or the foreign antigen in vaccine inoculation is different from the classical MHC- class I molecular presenting pathway (antigen presenting in the antigen presenting cell). Therefore, the mechanism of antigen degradation and peptide transport is likely to be significantly different in the above antigen presentation. In recent years, due to the recognition of the important role of cross-presentation in vaccination, its mechanism has attracted wide attention. The transport of endocytic proteins usually requires protein complexes containing Rab proteins, in which Rab proteins are key molecules of eukaryotic biofilm transport events. The human genome contains about 70 Rab and Rab like proteins, which are usually located in different membranous structural regions and regulate the four steps of membrane transport by guanosine acid dependent switching mechanism: vesicle budding and vesicle transport. The binding of vesicles and the fusion of vesicles with target membranes. Existing evidence suggests that several proteins are located in early sorting and return vesicles (Rab4,Rab5,Rab11,Rab18,Rab22 andRab25), while Rab7 and Rab9 are located in late endocysts and are now present in dendritic cells. There is no related study on Rab molecule, so we try to do this research in dendritic cells. The aim of this study was to investigate the expression of Rab5,Rab7 and their mutants in dendritic cells by transfection of Rab5 and Rab7 mutants, and to observe the Rab molecules associated with vesicular transport. Therefore, the cross-presentation of antigen occurred in the stage of phagocytic maturation. The effects of the two proteins on the cross-presentation of antigens were observed by using the inhibitory mutants Rab5S34N of Rab5 and Rab7T22N of Rab7. The results showed that Rab5S34N inhibited the cross-presentation of antigens, but Rab7T22N had no effect on cross-presentation of antigens. In order to further analyze the role of Rab5 in this process, we constructed the activated mutant RabSQ79L, of Rab5. The results showed that the antigen-presenting effect of Rab5 was the same as that of Rab5 wild type, which indicated that the early endocytosis was the main site of antigen cross-presentation. The results laid a foundation for further analysis of vesicle transport and regulation during antigen cross-presentation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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