Annexin A2蛋白和TNFα对新生隐球菌与宿主相互作用调控机制研究

发布时间：2018-06-05 05:26

本文选题：新生隐球菌 + 膜联蛋白A2　；参考：《第二军医大学》2017年博士论文

【摘要】：【研究目的】新生隐球菌是临床机会性致病真菌,能够突破宿主血脑屏障而感染中枢神经系统引起高致死率的隐球菌脑膜脑炎;隐球菌经呼吸道感染,首先定植于肺部,因此宿主肺部免疫反应强弱一定程度上决定隐球菌是否能够在体内增殖及扩散入血而造成中枢神经系统播散。本研究拟以隐球菌的致病机制为中心,主要围绕上述隐球菌感染机体过程中的两大问题,即隐球菌如何突破宿主血脑屏障、宿主针对隐球菌感染的肺部免疫反应的调节展开:第一部分主要探究宿主膜联蛋白家族中最重要的钙调节蛋白-膜联蛋白A2(Annexin A2,Anx A2)在隐球菌突破血脑屏障过程中的调节作用及其分子机制,旨在揭示隐球菌与宿主脑微血管内皮细胞相互作用过程中调节骨架蛋白重构相关的信号转导通路;第二、三部分主要研究构建表达宿主源性肿瘤坏死因子TNF?的转基因隐球菌菌株及通过该菌株诱导宿主产生保护性免疫反应的可行性,旨在探究感染灶局部应用TNF?的对隐球菌免疫调节效应,探索外源性细胞因子辅助治疗隐球菌感染的新途径。【研究方法】第一部分以体外血脑屏障模型、隐球菌-内皮细胞粘附模型为基础,通过慢病毒转染shRNA下调表达Anx A2蛋白,以探究其在隐球菌与内皮细胞粘附、穿越血脑屏障模型过程中的作用;通过探究Anx A2主要磷酸化位点磷酸化状态、构建去磷酸化细胞系、应用特异性抑制剂等,明确其与下游骨架调节蛋白的关系。第二部分通过PCR构建鼠源TNF?表达元件,以标准株H99为背景,借助基因枪转化系统构建表达鼠源性TNF?的新生隐球菌菌株,通过抗生素筛选、表型验证、细胞因子表达检测等获得能够稳定表达鼠源性TNFa的隐球菌菌株。第三部分以隐球菌吸入感染小鼠模型为基础,通过比较感染表达TNF?的转基因菌株与标准株后小鼠生存时间、不同感染时间点各脏器菌落负荷及肺组织病理等判断表达TNF?的转基因菌株是否具有保护作用;分离感染后小鼠肺组织白细胞,以特异性抗体标记白细胞表面抗原,应用流式细胞仪分析比较不同菌株感染后不同时间点白细胞类群和细胞数目变化;检测细胞培养上清中细胞因子表达谱遍化、巨噬细胞激活模式变化,明确表达TNF?的转基因菌株感染后对宿主免疫系统的调节作用。【结果】第一部分利用小鼠脑微血管内皮细胞系构建的体外血脑屏障模型符合研究要求;隐球菌刺激脑微血管内皮细胞引起AnxA2蛋白表达上调并改变其空间分布;封闭细胞膜外表面AnxA2后新生隐球菌穿越血脑屏障效率显著下降;下调内皮细胞AnxA2蛋白表达导致隐球菌与内皮细胞粘附率增加但不影响其穿越体外血脑屏障的整体效率;AnxA2缺失导致S100A10降解,进而引起隐球菌穿越血脑屏障效率下降;AnxA2 N末端23位酪氨酸(Tyr23)磷酸化调控下游Cofilin蛋白磷酸化而调节隐球菌穿越血脑屏障。第二部分通过基因枪转化获得的阳性克隆子具有TNF?表达序列;编号为T16的阳性克隆菌株在体外培养基体内环境条件下均能稳定表达、分泌TNFa且该菌株与标准株H99相比,无明显生长缺陷和毒力因子缺陷。第三部分T16菌株感染小鼠较野生株感染组生存时间明显延长、肺部菌荷量明显下降。肺部病理切片显示,T16菌株感染小鼠肺部隐球菌感染灶较野生株感染组病灶更为局限并由局部浸润的大量免疫细胞包裹。在固有免疫阶段,T16菌株分泌的TNF?促进宿主巨噬细胞通过经典激活方式激活(M1)提高了巨噬细胞杀菌能力;在适应性免疫阶段,免疫细胞类群比例分析显示T16菌株感染组小鼠肺部以诱导保护性T细胞浸润为主,非保护性嗜酸粒细胞浸润减少。同时,肺部非保护性Th2型细胞因子表达减少。T16菌株产生的TNF?诱导宿主肺组织中树突状细胞CD103+亚群和单核细胞成熟分化,是T16菌株诱导机体产生保护性适应性免疫应答的重要机制。【结论】第一部分宿主膜联蛋白AnxA2蛋白在隐球菌穿越血脑屏障的过程中发挥多重调节作用:一方面通过去磷酸化引起下游骨架调节蛋白激活,促进隐球菌与脑微血管内皮细胞粘附;另一方面,隐球菌穿越血脑屏障过程依赖AnxA2的伴侣蛋白S100A10,而AnxA2在该过程中发挥稳定S100A10的作用,从而间接调节隐球菌穿越血脑屏障的过程。第二、三部分表达鼠源性TNF?的新生隐球菌菌株T16分泌的TNF?在小鼠感染模型中能够诱导机体产生一定的保护性免疫应答,包括固有免疫阶段增强巨噬细胞杀菌能力,在适应性免疫阶段诱导免疫反应向保护性的I型免疫反应极化,为临床应用细胞因子辅助治疗隐球菌感染提供了新的思路。
[Abstract]:[Objective] Cryptococcus neoformans is a clinical opportunistic pathogenic fungus, which can break through the blood brain barrier of the host and infect the high lethal rate of Cryptococcus meningoencephalitis in the central nervous system. Cryptococcus neoformans is infected by respiratory tract and is first fixed in the lungs. Therefore, the strong weak immune response of the host lung determines whether Cryptococcus can be in body to some extent. This study intends to focus on the pathogenesis of Cryptococcus neoformans, focusing mainly on the two major problems in the process of cryptococcus infection, namely, how Cryptococcus breaks through the host blood brain barrier and the host's regulation of the immune response to cryptococcus infection: the first part of the study The most important calcium regulatory protein A2 (Annexin A2, Anx A2) in the host membrane protein family is to regulate the regulation and molecular mechanism of Cryptococcus neoformans in the process of breaking through the blood brain barrier. The aim is to reveal the signal transduction pathway related to the remodeling of the skeleton protein during the interaction of Cryptococcus neoformans and the host brain microvascular endothelial cells; second, The three part mainly studies the construction of transgenic Cryptococcus neoformans expressing the host derived tumor necrosis factor TNF and the feasibility of inducing protective immune response by this strain. The aim of this study is to explore the immunoregulation effect of TNF? On Cryptococcus neoformans and explore the new way of exogenous cytokines to treat cryptococcus infection. [method] [method] the first part is based on an in vitro blood brain barrier model, cryptococcal endothelial cell adhesion model, and down regulation of Anx A2 protein by lentivirus transfection to shRNA in order to explore its role in Cryptococcus neoformans adhesion to endothelial cells and through the process of crossing the blood brain barrier model, by exploring the phosphorylation of the main phosphorylation sites of Anx A2. State, constructing dephosphorylated cell lines, using specific inhibitors, and so on, to clarify the relationship with the downstream cytoskeleton regulation proteins. Second part of the mouse source TNF? Expression element was constructed by PCR, and the standard strain H99 was used as the background to construct a new Cryptococcus neoformans expressing mouse derived TNF with the aid of the gene gun transformation system. In the third part, the third part is based on the mouse model of Cryptococcus inhalation infection, and the expression of TNF by comparing the survival time of the transgenic strains expressing TNF with the standard strain, the drop load of the organs and the lung histopathology at different time points of the infection. Whether the transgenic strain has protective effect, the leukocyte in the lung tissue of the mice after infection is separated, the leukocyte surface antigen is marked with specific antibody, and the changes of the leucocyte group and the number of cells at different time points after the infection are analyzed by flow cytometry. The activation pattern changes clearly to express the regulatory effect of TNF? Transgenic strains infected with the host immune system. [results] the first part of the mouse brain microvascular endothelial cell line constructed in vitro blood brain barrier model conforms to the research requirements; Cryptococcus neoformans stimulates the brain microvascular endothelial cells to increase the expression of AnxA2 protein and change its empty space. The efficiency of Cryptococcus neoformans traversing the blood brain barrier after AnxA2 closed cell surface was significantly decreased, and the expression of AnxA2 protein in endothelial cells decreased the overall efficiency of Cryptococcus neoformans and endothelial cells, but did not affect the overall efficiency of the blood brain barrier across the body, and the AnxA2 deletion led to the degradation of S100A10, which could lead to Cryptococcus neoformans crossing the blood brain barrier. The efficiency of AnxA2 N terminal 23 tyrosine (Tyr23) phosphorylation regulates the phosphorylation of the downstream Cofilin protein to regulate Cryptococcus neoformans through the blood brain barrier. The positive clones obtained through the transformation of the second part of the gene gun have TNF? Expression sequence; the positive clones numbered as T16 can be expressed stably under the environment conditions of the culture medium in vitro. There was no obvious growth defect and virulence factor deficiency compared with the standard strain H99. The survival time of the third T16 strains infected mice was significantly longer than that of the wild strain infected mice, and the pulmonary bacteria charge decreased significantly. The lung pathological sections showed that the lung cryptococcus infection foci in the lungs of the T16 strain infected mice were more than those of the wild strain infected group. Localized and localized infiltration of immune cells. In the innate immune phase, the TNF secreted by T16 strain promotes macrophage bactericidal ability through classical activation (M1). In the adaptive immunization stage, the immune cell group ratio analysis shows that the T16 strain infected mice lung to induce protective T. Cell infiltration and non protective eosinophil infiltration decrease. Meanwhile, the expression of non protective Th2 type cytokines in the lung reduces the TNF produced by.T16 strain, and induces the maturation and differentiation of dendritic cells CD103+ subsets and monocytes in the host lung tissue, which is an important mechanism for the T16 strain to induce the protective adaptive immune response in the body. In part 1, the first part of the host membrane protein AnxA2 protein plays multiple regulatory roles in the process of Cryptococcus neoformans through the blood brain barrier: on one hand, the activation of the downstream cytoskeleton through dephosphorylation and the adhesion of Cryptococcus neoformans to the cerebral microvascular endothelial cells; on the other hand, Cryptococcus neoformans through the blood brain barrier depend on the chaperone protein of the AnxA2 S100A10, and AnxA2 plays the role of stabilizing S100A10 in this process, which indirectly regulates the process of Cryptococcus neoformans crossing the blood brain barrier. The second, third part expresses TNF secreted by Cryptococcus neoformans strain T16 of mouse derived TNF? In the mouse infection model, it can induce a certain protective immune response in the mouse infection model, including the enhancement of the inherent immune phase. The bactericidal ability of macrophages, inducing immune response to protective I type immunoreaction during adaptive immunization stage, provides a new idea for the clinical application of cytokines to treat cryptococcus infection.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R519.4

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