TGF-β对小鼠新生隐球菌感染及巨噬细胞功能影响的研究

发布时间：2018-01-15 17:34

本文关键词：TGF-β对小鼠新生隐球菌感染及巨噬细胞功能影响的研究　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景新生隐球菌是一种机会致病真菌,主要感染免疫缺陷人群,如AIDS/HIV患者、肿瘤病人和长期使用免疫抑制剂的群体。新生隐球菌菌体或孢子通常经呼吸道进入患者体内,造成肺部原发感染,进而通过血脑屏障进入中枢神经系统,引起致命性隐球菌性脑膜炎。机体感染隐球菌的结局极大程度上取决于机体的免疫状态,Th1免疫反应占主要时可清除隐球菌。肺巨噬细胞是机体抵御隐球菌的第一类免疫细胞,可通过内吞和氧化杀伤方式在第一时间抵抗入侵的隐球菌,同时巨噬细胞也是连接固有免疫和适应性免疫的关键环节,可募集T细胞至感染灶,形成细胞因子微环境,进而调控后续的获得性免疫反应。巨噬细胞在机体抗新生隐球菌免疫反应中扮演重要角色。转化生长因子(TGF-β)是一种多效细胞因子,在调节免疫反应方面有重要作用,可影响免疫细胞增殖、分化及相应功能,诱导免疫耐受和影响Th1/Th2细胞平衡。本课题将研究TGF-β对巨噬细胞吞噬、杀伤功能的影响,以及在机体抗隐球菌免疫方面的作用。研究目的本课题旨在研究TGF-β对巨噬细胞功能的影响,以及TGF-β在机体抗隐球菌感染方面的作用。研究方法用重组小鼠TGF-β提前干预J774A.1巨噬细胞,随后用新生隐球菌感染巨噬细胞,观察巨噬细胞吞噬率的改变;予以TGF-β干预隐球菌感染后巨噬细胞,分别比较巨噬细胞内、外隐球菌菌落负荷在TGF-β干预后变化,用以评价巨噬细胞对胞内、外隐球菌杀伤能力;为进一步研究巨噬细胞杀伤能力改变,用溶菌酶试剂盒检测巨噬细胞溶菌酶的分泌量,用Griess reagent试剂盒检测巨噬细胞NO生成量,并比较TGF-β干预后二者生成量的变化;建立小鼠隐球菌吸入感染模型,将重组小鼠TGF-β经尾静脉注射干预小鼠,分两组在感染早期和晚期分别干预,观察小鼠生存曲线、肺、脑组织菌落负荷,以及肺、脑、外周血中Th1/Th2/Th17细胞因子浓度变化。结果TGF-β干预导致巨噬细胞吞噬率下降,而巨噬细胞NO生成量和溶菌酶分泌量均上升,对吞噬入胞内和胞外的隐球菌杀伤力均增加。在小鼠吸入感染模型,进行生存曲线分析,TGF-β早期干预组小鼠抵抗隐球菌能力减弱,死亡时间提前。此外,感染早期予TGF-β干预组,肺部菌落负荷增加,晚期隐球菌播散入脑数量增加,肺部IFN-γ、TNF-α浓度降低,IL-6浓度增高,脑组织和外周血Th细胞因子未见变化。感染后期予TGF-β干预小鼠组,肺部菌落下降,脑部隐球菌量无差异,肺组织中TNF-α和IL-6浓度升高,脑组织和外周血Th细胞因子亦未见变化。结论TGF-β抑制巨噬细胞吞噬隐球菌能力,但可通过增加溶菌酶和NO的生成量,提升巨噬细胞对隐球菌的杀伤能力。在小鼠吸入隐球菌感染模型,早期TGF-β干预将抑制巨噬细胞吞噬作用,进而抑制有效炎症反应,小鼠抵抗隐球菌能力下降,导致隐球菌播散,死亡时间提前。晚期TGF-β干预可提高巨噬细胞杀伤功能,同时可提高肺部TNF-α浓度,增强抗隐球菌能力,肺部菌落负荷量下降,有助于使感染局限化。
[Abstract]:The research background of Cryptococcus neoformans is an opportunistic fungal infection in immunocompromised population, mainly, such as AIDS/HIV patients, cancer patients and long-term use of immunosuppressant group. Cryptococcus neoformans mycelium or spores are usually caused by respiratory tract in patients, primary lung infection, and then enter the central nervous system through the blood-brain barrier, causing fatal hidden neoformans meningitis. The immune status of cryptococcal infection outcome greatly depends on the body's immune response, Th1 accounted for the major may remove Cryptococcus. Pulmonary macrophage is the first class of immune cells against Cryptococcus neoformans can, through endocytosis and oxidative killing ways to resist the invasion in the first time, and it is also a key link connect the innate and adaptive immune and macrophage, can induce T cells to form foci of infection, cytokine microenvironment, and subsequent control The acquired immune response. Macrophages play an important role in the new body cryptococcal immune response. Transforming growth factor (TGF-) is a pleiotropic cytokine that plays an important role in the regulation of immune response, can affect the immune cell proliferation, differentiation and the corresponding function, the induction of immune tolerance and influence the balance of Th1/Th2 cells. This paper will study TGF- beta on macrophage phagocytosis, cytotoxicity function, and the role in the anti Cryptococcus immunity. The purpose of this study is to study the effect of TGF- on macrophage function beta, beta and TGF- in anti cryptococcus infection and the role of research methods with recombinant mouse J774A.1 macrophage TGF- beta in early intervention then, with Cryptococcus neoformans infection of macrophages, the phagocytosis rate of macrophage change; TGF- beta cryptococcal infection after intervention were compared with macrophages, macrophages Intracellular external cryptococcal colony load changes in the intervention of TGF- beta, evaluated by macrophages, and cryptococcal killing ability; for the further study of macrophage killing ability changes with the secretion of macrophages lysozyme lysozyme detection kit for detection of Griess, macrophage NO reagent kit generation, and compare the changes of beta TGF- the two generation of intervention; cryptococcal infection model of mice inhaled, the recombinant mouse beta TGF- after intravenous intervention mice were divided into two groups of intervention in the early and late infection, observe the survival curves of mice, lung, brain, lung, brain colony load, changes of Th1/Th2/Th17 cytokines in peripheral blood results. TGF- beta intervention led to the phagocytosis rate of macrophages decreased, and the production of NO and lysozyme secretion increased, increased the phagocytosis of Cryptococcus bacteria lethality into intracellular and extracellular. In the mouse model of infection by inhalation, survival curve analysis, early intervention group TGF- beta mice against Cryptococcus neoformans ability is abate, time of death in advance. In addition, the early stage of infection with TGF- beta intervention group, lung colony load increases, the late cryptococcal spread into the brain by increasing the number of lung IFN- gamma, alpha TNF- concentration decreased, the concentration of IL-6 increased brain tissue and peripheral blood Th cell factor did not change. The late stage of infection with TGF- beta treated mice group, lung colonies decreased, no difference in brain Cryptococcus, increased TNF- alpha and IL-6 concentration in lung tissue, brain tissue and peripheral blood Th cells was also no change. Conclusion TGF- beta inhibits macrophage phagocytosis of Cryptococcus neoformans ability, but increased the production of lysozyme and NO, enhance the killing capacity of macrophages of Cryptococcus neoformans in mice model. Inhalation of cryptococcal infection, early intervention of TGF- beta will inhibit macrophage phagocytosis by inhibiting, and Effect of inflammatory reaction in mice against Cryptococcus neoformans resulting in decreased ability to spread, the time of death in advance. The late TGF- beta intervention can improve macrophage killing function, also can improve the lung TNF- concentration, enhance the anti Cryptococcus ability, lung colony load decreased, helps to make localized infection.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R519.4

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