Rac2负性调节树突状细胞的交叉递呈

发布时间：2018-08-08 15:30

【摘要】： 树突状细胞(dendritic cells,DC)是主要的一种抗原递呈细胞,目前认为DC细胞抗原递呈的方式有三种:一种是DC细胞吞噬外来抗原,并加工处理形成表位肽,与MHCⅡ类分子结合,从而激活CD4+T细胞;另外一种指DC细胞将内源性合成的蛋白处理与MHCⅠ类分子结合激活CD8+T细胞;Bevan提出了一种新的DC细胞递呈抗原的方式——交叉递呈(cross presentation),它是指抗原递呈细胞将外源性的抗原加工处理,负载到MHCⅠ类分子表面形成复合物,从而激活初始CD8+T细胞引起细胞免疫反应的过程。交叉递呈对于监视组织中的肿瘤以及清除那些不能感染抗原递呈细胞的病毒起着十分重要的作用。详细了解DC细胞交叉递呈的机制将有助于我们更好的找到抗肿瘤和抗病毒的途径,为肿瘤和病毒感染的免疫治疗提供新的线索。但是目前对于DC细胞交叉递呈的调节机制研究甚少。以往许多研究均表明Rho GTPase参与调节DC细胞生理活动的许多方面,包括细胞形态的重塑,树突的伸展,抗原的吞噬以及T细胞的活化。Cdc42和Rac1影响DC细胞对外来抗原的吞噬能力,其中Cdc42的活化对于DC细胞成熟过程中吞噬能力的变化起着重要作用;RhoB影响LPS刺激后DC细胞表面MHCⅡ分子的表达;另外,Rac1抑制性突变体的转基因小鼠DC细胞吞噬凋亡细胞的能力下降,从而影响交叉递呈。所以我们希望了解Rho家族蛋白在交叉递呈中扮演什么样的角色,是什么样的机制参与其中? 目的:本研究希望通过一系列的实验筛选出对交叉递呈有作用的Rho蛋白分子并进一步探讨可能的机制。方法: 1、利用RNAi干扰的方法筛选常见的六种Rho分子:RNAi筛选的方法有利于我们将不同的分子进行水平比较,并且为了解不同细胞的生物学功能提供线索。利用合成的siRNA,本研究分别干扰DC细胞中六种常见的Rho分子,然后利用T细胞杂交瘤B3Z检测干扰后DC细胞交叉递呈的能力;并且通过实时定量PCR的方法验证了RNA干扰的效率; 2、表达Rac2不同性质突变体的DC细胞交叉递呈能力分析:根据Rho分子的特性,活化型突变体和抑制型突变体常常作为研究Rho分子功能的有效手段。利用点突变的方法,本研究构建了Rac2的两种突变体——活化型突变体Rac2Q61L和抑制型突变体Rac2D57N,并且与黄色荧光蛋白EYFP融合表达;通过慢病毒载体高效感染树突状细胞,得到稳定表达EYFP-Rac2突变体的DC2.4,然后通过ELISA检测表达不同突变体的DC细胞交叉递呈的能力; 3、分子机制探讨: (1)吞噬过程中Rac2的活化:荧光共振能量转移(F?rster Resonance Energy Transfer, FRET)是目前研究两蛋白分子之间相互作用的最有效的手段。相对于共聚焦显微镜,它不但表明两分子之间空间距离的相近,更能从功能方面阐明两分子之间的关系。由于Rac2活化后可以与下游的PAK分子相互作用,本研究利用慢病毒载体,将DC细胞同时转染黄色荧光蛋白标记的Rac2以及红色荧光蛋白标记的PAK分子,利用激光共聚焦显微镜观察在吞噬外来抗原的过程中Rac2分子的活化情况以及在细胞中的定位; (2)吞噬实验:利用表达不同Rac2突变体的DC细胞,观察转染不同突变体的DC细胞对外来的表达红色荧光蛋白的细菌抗原在不同时相的吞噬能力,了解Rac2分子对吞噬功能的影响; (3)内源性递呈实验:这种DC细胞可以内源性表达OVA蛋白,有效地将表位肽与MHCⅠ类分子复合物递呈到DC细胞表面,然后用表达EYFP-Rac2突变体的慢病毒感染DC-OVA细胞,观察Rac2对DC-OVA细胞内源性递呈的影响,说明Rac2分子在DC细胞交叉递呈中影响环节; 4、Cd11c-EYFP-Rac2Q61L转基因小鼠的建立:为了深入探讨和观察Rac2分子对DC细胞的功能影响,我们通过能在DC细胞中特异性表达EYFP-Rac2Q61L的慢病毒载体来感染小鼠胚胎细胞,得到了Cd11c-EYFP-Rac2Q61L转基因小鼠,该实验平台的建立为我们研究交叉递呈相关分子提供了有效的手段,从而便于我们可以从在体水平检测该分子对交叉递呈能力的影响。结论:通过本研究,我们观察到:(1)不同的Rho分子对DC细胞交叉递呈的能力有所不同,干扰Rac2表达的DC细胞交叉递呈能力明显上升;(2)通过构建慢病毒载体表达活化型和抑制型Rac2突变体,并将其转染DC细胞,我们发现过度表达活化型突变体Rac2Q61L可以有效降低DC细胞的交叉递呈;(3)在吞噬外来抗原的过程中,Rac2的活化发生在吞噬外来抗原的胞膜部分,活化下游PAK分子;(4)Rac2不影响DC细胞吞噬外来抗原的量以及内源性递呈。可以初步得到结论:Rac2不同于其它的Rho分子,它负性调节树突状细胞的交叉递呈,并且Rac2的活化主要发生在胞膜周围,通过与PAK分子的相互作用调节DC细胞的后续过程,可能与吞噬体的酸化,抗原降解有关而不是作用于交叉递呈的其余阶段。本研究为深入了解树突状细胞交叉递呈的分子机制奠定了基础,而且这种负性调节分子Rac2的发现为了解疾病状态下如肿瘤局部的免疫耐受提供了新的线索。
[Abstract]:Dendritic cells (DC) is one of the main antigen presenting cells. Currently, there are three types of antigen presenting DC cell antigens: one is that DC cells phagocytic external antigens, and processing and processing the epitope peptide to combine with MHC class II molecules to activate CD4+T cells; the other means that DC cells process endogenous synthetic proteins with M. HC class I molecules combine to activate CD8+T cells; Bevan has proposed a new way of DC cell presenting antigen, cross presentation (cross presentation), which refers to the processing of exogenous antigen by antigen presenting cells, which is loaded to the surface of MHC class I molecules to form complex, activating the initial CD8+T cells to cause cellular immune response. A detailed understanding of the mechanism of the cross delivery of DC cells will help us better find ways to resist cancer and antivirus, and provide new clues for the immunotherapy of cancer and disease. At present, there is little research on the regulation mechanism of DC cell cross delivery.
Many previous studies have shown that Rho GTPase participates in many aspects of regulating physiological activity of DC cells, including remodeling of cell morphology, extension of dendrites, phagocytosis of antigen, and the activation of T cells by.Cdc42 and Rac1 in the phagocytosis of DC cells to foreign antigens, in which the activation of Cdc42 plays a role in the change of phagocytosis in the process of DC cell maturation. RhoB affects the expression of MHC II molecules on the surface of DC cells after LPS stimulation; in addition, the ability of DC cells to phagocytic apoptotic cells in the Rac1 inhibited mutant mice decreases, thus affecting the cross presentation. So we want to know what role the Rho family protein plays in the cross presentation, and what kind of mechanism is involved. Among them?
OBJECTIVE: In this study, we hope to screen out the Rho protein molecule which has effect on cross-presentation through a series of experiments and further explore the possible mechanism.
Method:
1, using the RNAi interference method to screen six common Rho molecules: the RNAi screening method helps us to compare different molecules and provide clues for understanding the biological functions of different cells. Using synthetic siRNA, this study interferes with six common Rho molecules in DC cells, and then uses T cell hybridoma B3Z detection. The ability of DC cells to cross presentation was measured after interference, and the efficiency of RNA interference was verified by real-time quantitative PCR.
2, expressing the cross presenting ability of DC cells with different properties of Rac2: according to the characteristics of Rho molecules, active and inhibitory mutants are often used as an effective means to study the function of Rho molecules. By using point mutation methods, this study constructed two kinds of mutants of Rac2, active mutant Rac2Q61L and inhibition type mutation. The body Rac2D57N was fused with the yellow fluorescent protein EYFP, and the DC2.4 was expressed stably by the high efficient infection of the dendritic cells by the lentivirus vector, and then the ability to express the cross delivery of the DC cells with different mutants was detected by ELISA.
3, the discussion of molecular mechanism:
(1) activation of Rac2 during the phagocytosis: the fluorescence resonance energy transfer (F? Rster Resonance Energy Transfer, FRET) is the most effective means to study the interaction between the two protein molecules. Compared with confocal microscopy, it not only indicates the proximity of the spatial distance between the two molecules, but also clarifies the relationship between the two molecules from the functional aspect. Since Rac2 can interact with the downstream PAK molecules after activation, this study uses the lentivirus vector to simultaneously transfect the DC cells to the Rac2 labeled with the yellow fluorescent protein and the PAK molecules marked by the red fluorescent protein. The activation of the Rac2 molecules in the process of phagocytic antigen is observed by laser confocal microscopy and in the cell. Positioning;
(2) phagocytosis experiment: using DC cells expressing different Rac2 mutants to observe the phagocytic ability of DC cells transfected with different mutants to different phase, and to understand the effect of Rac2 molecules on phagocytosis.
(3) endogenous presentation experiment: the DC cells can express the OVA protein endogenous, effectively transfer the epitope and MHC class I complex to the DC cell surface, and then use the EYFP-Rac2 mutant of the lentivirus to infect the DC-OVA cells, and observe the effect of Rac2 on the endogenous recurrence of DC-OVA cells, indicating that the Rac2 molecules are in the cross delivery of DC cells. Influence link;
4, the establishment of Cd11c-EYFP-Rac2Q61L transgenic mice: in order to explore and observe the effect of Rac2 molecules on the function of DC cells, we infect mouse embryo cells by expressing EYFP-Rac2Q61L lentivirus vector specifically in DC cells, and obtained the Cd11c-EYFP-Rac2Q61L transgenic mice. The cross-presentation-related molecules provide an effective means for us to detect their effects on cross-presentation at the in vivo level.
Conclusion: through this study, we observed: (1) the ability of different Rho molecules to cross delivery of DC cells was different, and the cross delivery ability of DC cells that interfered with Rac2 expression increased obviously. (2) the expression of active and inhibitory Rac2 mutants by the construction of lentivirus vector and transfected to DC cells, we found overexpression of activation mutation. Body Rac2Q61L can effectively reduce the cross delivery of DC cells; (3) in the process of phagocytic antigen, the activation of Rac2 occurs in the membrane part of the external antigen, activating the downstream PAK molecule; (4) Rac2 does not affect the amount of the foreign antigen phagocytic and the endogenous delivery.
It can be concluded preliminarily that Rac2 is different from other Rho molecules, which negatively regulates the cross delivery of dendritic cells, and the activation of Rac2 mainly occurs around the cell membrane. Through the interaction with PAK molecules, the subsequent process of DC cells may be related to the acidification of the phagocytic, antigenic degradation, and not the rest of the DC cells. This study provides a basis for understanding the molecular mechanism of dendritic cells cross presenting, and the discovery of this negative regulatory molecule, Rac2, provides a new clue to understanding the local immune tolerance in the disease.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【相似文献】