CPAF、STING和MyD88在鼠型衣原体生殖道感染中的作用研究

发布时间：2018-03-26 09:50

  本文选题：衣原体　切入点：CPAF　出处：《南方医科大学》2017年博士论文

【摘要】：沙眼衣原体(Chiamydia trachomatis,Ct)是常见性病病原体之一,是一类活细胞内寄生原核生物。入侵宿主后释放最重要的蛋白因子:衣原体蛋白酶样激活因子(Chlamydial Protease-Like Activity Factor,CPAF),致病中发挥重要作用。干扰素基因刺激因子(stimulator of interferon genes,STING)和髓样分化因子88(myeloiddifferentiationfactor88,MyD88)都是胞内天然免疫反应信号通路中的重要接头蛋白。两者借助共同下游信号通路与衣原体感染相关,可能在其致病过程中发挥重要作用。鼠衣原体(Chiamydia muridarum,mouse pneumonitis agent,MoPn)基因结构和组成与人型Ct极为相似。因此,用MoPn建模研究Ct致病机制。目的:本研究体外验证CPAF可能协助MoPn逃避胞外免疫监视,进而选STING-/-、MyD88-/-和C57BL/6J小鼠为对象,分别通过不同的MoPn感染途径构建动物模型,检测生殖道不同组织段的MoPn播散量、阴道清除速度、病理变化,分析STING、MyD88在生殖道功能屏障中的作用,研究衣原体可能的致病机制。方法:(1)用T细胞、DC及OT1或OT2抗原模拟抗原递呈过程,与不同浓度CPAF作用,ELISA分别检测上清液中IL-2的量。电泳分离及卡马斯亮蓝法,分析CPAF处理及未处理衣原体抗原组电泳条带变化。分别选一种能被CPAF酶切及未能酶切的衣原体抗原代替OT2及OT1,重复抗原递呈过程。ELISA分别检测上清液IL-2的量。(2)用2X105IFUs MoPn经不同途径(阴道、宫腔、子宫远端段及卵巢囊)感染野生型、STING-/-、MyD88-/-小鼠。不同时间点处死小鼠,一部分生殖道大体评分、HE染色、组织和细胞免疫荧光技术(immunofluorescence,IF),对比组织病变程度。(3)一部分小鼠生殖道分段匀浆,IF检测子宫-输卵管屏障(uterotubal junction,U-T屏障)及宫颈屏障两侧衣原体量的差别。结果:1.CPAF选择性抑制MHCⅡ-OT2抗原递呈过程而非MHCⅠ-OT1(***P0.001)。CPAF同样具有选择性酶切衣原体T细胞抗原(***p0.001)。2.经宫颈感染,STING-/-小鼠上生殖道大体病变和显微镜下炎症评分比野生型更严重(**p0.01)。提示,STING可能参与U-T屏障功能。3.经宫颈感染,MyD88-/-小鼠带菌时间明显延长(**P0.01)。经宫颈及阴道感染均显示,MyD88-/-小鼠上生殖道组织大体和微观病变更严重(**P0.01)。提示,宫颈屏障及U-T屏障功能都可能因MyD88缺失而受影响。4.经卵巢囊及子宫远端段感染,C57BL/6J小鼠U-T屏障两侧衣原体量有显著差异(*p0.05),STING-/-及MyD88-/-差异不显著(p0.05)。提示两者参与U-T结构屏障功能。结论:1)CPAF选择性酶切Ct抗原,协助Ct逃避宿主胞外免疫监视。2)STING信号通路主要通过参与U-T屏障功能,控制衣原体上行感染输卵管。3)MyD88信号通路同时参与U-T屏障和宫颈屏障功能,抑制衣原体播散,控制衣原体上行感染输卵管。
[Abstract]:Chlamydia trachomatisme (chlamydia trachomatis) is one of the common pathogens of STDs. It is a kind of living cell parasitic prokaryote. The most important protein factor released after invading the host is Chlamydial Protease-Like Activity Factorus, which plays an important role in pathogenesis. Interferon gene stimulator of interferon genes-STINGA) and medulla. Like differentiation factor 88 myeloid differentiation factor 88 (MyD88) is an important junction protein in the intracellular innate immune response signaling pathway, both of which are associated with chlamydia infection through a common downstream signaling pathway. The structure and composition of chlamydia muridarum muridarum muridarum mouse pneumonitis agentsMoPnnare very similar to those of human Ct. Objective: to study the mechanism of Ct pathogenicity by MoPn. Objective: to verify in vitro that CPAF may assist MoPn to escape from extracellular immune surveillance, and then select STING-P / P MyD88-r- and C57BL/6J mice to construct animal models through different MoPn infection pathways. The amount of MoPn spread, vaginal clearance speed, pathological changes in different tissue segments of the reproductive tract were measured. The role of STINGMI-MyD88 in the reproductive tract functional barrier was analyzed. To study the possible pathogenic mechanism of Chlamydia chlamydia. Methods T cell DC and OT1 or OT2 antigen mimic antigen presentation were used to detect the amount of IL-2 in supernatant by Elisa with different concentrations of CPAF. The electrophoretic bands of CPAF treated and untreated chlamydia chlamydia antigen groups were analyzed. A chlamydia antigen that could be digested by CPAF or not was selected to replace OT2 and OT1 respectively. The repeated antigen presentation process. Elisa was used to detect the amount of supernatant IL-2 with 2X105IFUs. MoPn goes through different ways (vagina, vagina, Uterine cavity, distal uterine segment and ovarian sac) were infected with wild type STING-P / -MyD88-r-mice. The mice were killed at different time points, and some of the reproductive tract gross scores were stained with HE. Tissue and cell immunofluorescence techniques were used to detect the difference of chlamydia content between uterotubal junctional U-T barrier and cervical barrier. Results: 1. CPAF selectivity. The inhibition of MHC 鈪，

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