甘露聚糖结合凝集素对T淋巴细胞活化、增殖及分化的调节作用及其在矽肺中的意义

发布时间：2017-12-31 12:26

本文关键词：甘露聚糖结合凝集素对T淋巴细胞活化、增殖及分化的调节作用及其在矽肺中的意义　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：MBL属于C型凝集素超家族中胶凝素(collectins)家族成员,是肝细胞分泌的血浆蛋白,对于抗御病原体感染和清除凋亡细胞及免疫复合物具有重要意义。我们近期研究发现MBL可调节人单核细胞(Mo)、树突状细胞(DC)等抗原提呈细胞的分化发育,然而,MBL对T淋巴细胞是否具有直接的调节作用,目前尚无相关文献报道。为进一步研究MBL对获得性免疫的调节作用,本课题以T细胞为切入点,初步探索MBL对T细胞活化、增殖、分化的影响及其分子机制。第一章MBL对T淋巴细胞活化增殖的调节作用及其机制随着天然免疫研究的深入,发现胶凝素家族的一些成员和获得性免疫之间的联系越来越紧密。研究表明表面活性物质-A(SP-A),表面活性物质-D(SP-D)可直接抑制T细胞增殖活化。MBL与SP-A、SP-D的同源性,尤其是其与SP-A在结构上的高度相似,我们推测MBL很有可能参与获得性免疫应答。我们使用MACS负选法分离人纯化T细胞,在CD3/CD28共刺激T细胞活化体系中加入血浆纯化MBL蛋白可减弱T细胞的活化与增殖,外源性的IL-2可以逆转MBL的作用;MBL抑制CD3/CD28共刺激T细胞活化过程中ZAP-70蛋白和活化T细胞接头蛋白及NF-kappa B及MAPK信号通路的活化。MBL还可使活化T细胞处于无应答状态。MBL可以结合活化T细胞而非初始T细胞;MBL通过其CLR结构域与活化T细胞表面的CRT蛋白直接结合,使用抗CRT抗体封闭T细胞表面的CRT蛋白,可以逆转MBL对T细胞的抑制作用。我们还发现与MBL结构高度同源的C1q及凝集素家族新成员CL-11具有与MBL同样的作用方式,即通过与活化T细胞表面CRT作用来抑制T细胞的增殖。总而言之,本部分研究确定了 MBL可通过其CLR结构域与活化T细胞表面CRT蛋白作用,这可能是MBL抑制T细胞活化与增殖的机制。第二章MBL对T淋巴细胞功能的调节作用及其在矽肺中的意义负向免疫调控机制对于维持体内免疫系统自稳和预防免疫系统过度活化具有重要作用。我们首次报道了矽肺患者血清中MBL浓度升高,且血清MBL浓度与CD45RO+T细胞比率呈负相关,与肺功能呈正相关;MBL亦能抑制矽肺病人T细胞活化与增殖;早期矽肺患者中MBL浓度与Th17细胞呈负相关,与Treg细胞呈正相关。我们亦发现MBL-L-小鼠对SiO2诱导的肺损伤较为敏感,表现为严重肺泡炎和肺水肿;与WT小鼠相比,MBL-/-小鼠受SiO2攻击后体内Th17细胞比率较高,Treg细胞比率较低,这可能与MBL-L-小鼠受致病因素攻击后,体内STAT3磷酸化水平较高有关。在体外Th17分化培养系统中,MBL可抑制人naive CD4+ T细胞向Th17分化,促进其向Treg分化,这可能与MBL抑制IL-6诱导的STAT3磷酸化有关。综上,本课题研究发现MBL可抑制T细胞的增殖、活化及相关细胞因子的分泌,并提出MBL可能通过调节T细胞的功能在矽肺中发挥免疫调节作用。本研究将为天然免疫及其指导获得性免疫应答的基础理论提供实验依据,并为MBL和T细胞功能紊乱有关疾病的诊治研究提供新的研究基础。
[Abstract]:MBL is a member of the C-type lectin superfamily, which is the plasma protein secreted by hepatocytes. We recently found that MBL can regulate human monocyte moths. The differentiation and development of antigen-presenting cells such as dendritic cells (DC) and other antigen-presenting cells, however, whether MBL has direct regulatory effect on T lymphocytes. In order to further study the role of MBL in the regulation of acquired immunity, T cells as a starting point, the preliminary exploration of MBL on T cell activation, proliferation. Chapter 1 the effect of MBL on T lymphocyte activation and proliferation and its mechanism with the development of innate immunity. A growing association between some members of the gelatin family and acquired immunity has been found. The surface active substance -DnSP-D directly inhibited the proliferation and activation of T cells. MBL was homologous to SP-An SP-D, especially its structure was similar to that of SP-A. We speculated that MBL might be involved in the acquired immune response. We used MACS negative selection method to isolate and purify human T cells. The activation and proliferation of T cells were inhibited by the addition of plasma purified MBL protein in CD3/CD28 costimulated T cell activation system, and the effect of MBL was reversed by exogenous IL-2. Inhibition of ZAP-70 protein, activated T cell junction protein and NF-kappa by MBL during CD3/CD28 costimulatory T cell activation. The activation of B and MAPK signaling pathways can also make activated T cells in a nonresponsive state. MBL can bind to activated T cells instead of initial T cells. MBL binds directly to the CRT protein on the activated T cell surface through its CLR domain and uses anti-#en3# antibody to block the CRT protein on the T cell surface. We also found that C1q and CL-11, a new member of the lectin family, which are highly homologous to the structure of MBL, have the same effect as MBL. That is, through the activation of T cell surface CRT to inhibit the proliferation of T cells. All in all, this part of the study confirmed that MBL through its CLR domain and activated T cell surface CRT protein interaction. This may be the mechanism by which MBL inhibits the activation and proliferation of T lymphocytes. Chapter 2 the regulatory effect of MBL on T lymphocyte function and its significance in silicosis. Prevention of excessive activation of the immune system plays an important role. We reported for the first time that the serum MBL level in silicosis patients was elevated. The concentration of serum MBL was negatively correlated with the ratio of CD45RO T cells and positively correlated with pulmonary function. MBL also inhibited the activation and proliferation of T cells in silicosis patients. The concentration of MBL in early silicosis patients was negatively correlated with Th17 cells and positively correlated with Treg cells. We also found that MBL-L- mice were more sensitive to SiO2 induced lung injury. Severe alveolitis and pulmonary edema; Compared with WT mice, the ratio of Th17 cells was higher in MBL-r-mice after being attacked by SiO2, which may be similar to that in MBL-L- mice after attack by pathogenic factors. The phosphorylation level of STAT3 in vivo was higher than that in vitro. In vitro Th17 differentiation culture system could inhibit the differentiation of human naive CD4 T cells into Th17. It may be related to the inhibition of STAT3 phosphorylation induced by IL-6 by MBL. In summary, we found that MBL can inhibit the proliferation of T cells. Activation and secretion of cytokines. It is suggested that MBL may play an immunomodulatory role in silicosis by regulating the function of T cells. This study will provide experimental basis for innate immunity and its basic theory of guiding acquired immune response. It also provides a new basis for the diagnosis and treatment of diseases related to MBL and T cell dysfunction.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R392

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