ICOSL介导的反向信号在调控GC成熟过程中的作用及机制研究

发布时间：2018-03-12 17:27

本文选题：GC　切入点：体液免疫　出处：《苏州大学》2016年博士论文　论文类型：学位论文

【摘要】：研究目的:生发中心(Germinal center,GC)辅助T细胞(Tfh或GC T)支持GC B细胞活化、存活以及功能成熟的分子机制还不是很清楚。迄今为止,GC B细胞上CD40接受Tfh细胞上CD40L的辅助信号是被研究最多的Tfh细胞提供的辅助信号。而GC B细胞内发生的许多现象又无法完全通过CD40的活化来解释。GC内T-B相互作用有众多的共刺激分子信号通路的参与,这些信号通路在GC内对B细胞的辅助作用还需要进一步的研究。我们想通过我们的研究,找出GC内对B细胞存活及功能成熟的新的分子机制,使我们对GC内的分子信号通路有更深入的了解。研究方法:分选GC B细胞以及GC T细胞,然后体外共培养,培养体系中加入已经报道的共刺激分子通路的封闭性抗体,先以GC B细胞的存活为指标,通过体外的筛选实验找出在GC阶段,哪些信号通路对GC B细胞存活的辅助起关键作用。然后再通过体外刺激,及基因敲除技术找出起关键作用的共刺激分子通路,及这些共刺激分子通路通过激活哪些信号通路来辅助GC B细胞的存活及功能成熟。最后通过基因敲除和骨髓重建等技术来证明相关的共刺激分子通路在体内的作用。成果和结论:我们首先通过体外的筛选实验证明在参与T-B互作的众多共刺激分子通路中,ICOS-ICOSL对GC B细胞的促存活作用最为关键。进一步的研究表明GC T细胞提供给GC B细胞的存活和功能成熟辅助信号是通过ICOS-ICOSL信号通路并且可通过ICOSL介导的反向信号来实现对GC B细胞存活的促进作用。然而,我们进一步发现GC B上CD40介导的信号对GC B的存活似乎并没有明显的作用。用ICOS重组蛋白静脉注射ICOS基因缺陷的小鼠能够部分恢复GC的形成和抗体产生。多种ICOSL突变小鼠,包括整个B细胞ICOSL完全缺失、GC B细胞ICOSL缺失及B细胞ICOSL胞内段缺失小鼠,都表现出体液免疫缺陷及GC形成受损。我们进一步发现GC B细胞上ICOSL介导的反向信号通过激活经典的NF-κB信号通路来上调跟GC B细胞存活和功能成熟相关的蛋白的表达,如MCL-1、c-MYC、FOXO1及AID等。因此,我们的实验数据表明ICOSL-ICOS信号通路在GC阶段起着双向的作用。
[Abstract]:Objective: Germinal Center GC) assisted T cell Tfh or GC T to support the activation of GC B cells. The molecular mechanism of survival and functional maturity is not well understood. Up to now, CD40 on GC B cells receives the auxiliary signal of CD40L on Tfh cells, which is provided by the most studied Tfh cells. Multiple phenomena can not be fully explained by the activation of CD40. The T-B interaction in GC is involved in many costimulatory molecular signaling pathways. The role of these signaling pathways in assisting B cells in GC requires further study. We want to find out new molecular mechanisms for the survival and functional maturity of B cells in GC. Methods: GC B cells and GC T cells were separated, then co-cultured in vitro, and the closed antibodies of the reported costimulatory molecular pathways were added to the culture system. The survival of GC B cells was used as an index, and in vitro screening experiments were conducted to find out which signaling pathways played a key role in assisting the survival of GC B cells, and then stimulated in vitro. And gene knockout techniques to identify key costimulatory molecular pathways, And which signaling pathways are activated to assist the survival and functional maturation of GC B cells. Finally, gene knockout and bone marrow reconstruction techniques are used to prove the role of costimulatory molecular pathways in vivo. Results and conclusions: we first demonstrated that among the numerous co-stimulatory molecular pathways involved in T-B interaction, ICOS-ICOSL is the most important factor in promoting the survival of GC B cells. Further studies have shown that GC T cells can promote the survival of GC B cells. The auxiliary signal of survival and functional maturation of GC B cells is enhanced by ICOS-ICOSL signaling pathway and reverse signal mediated by ICOSL. However, We further found that the signal mediated by CD40 on GC B did not seem to have a significant effect on the survival of GC B. mice with ICOS gene deficiency injected with ICOS recombinant protein could partially restore GC formation and antibody production. Including the complete deletion of ICOSL in the whole B cell and the deletion of ICOSL in the GC B cell and the deletion of the intracellular segment of the ICOSL in the B cell. We further found that the reverse signal mediated by ICOSL on GC B cells upregulated the expression of proteins related to survival and functional maturation of GC B cells by activating the classical NF- 魏 B signaling pathway. Therefore, our experimental data show that the ICOSL-ICOS signaling pathway plays a bidirectional role in the GC phase.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

【相似文献】