本文選題：衣原體　切入點(diǎn)：CPAF　出處：《南方醫(yī)科大學(xué)》2017年博士論文

【摘要】：沙眼衣原體(Chiamydia trachomatis,Ct)是常見性病病原體之一,是一類活細(xì)胞內(nèi)寄生原核生物。入侵宿主后釋放最重要的蛋白因子:衣原體蛋白酶樣激活因子(Chlamydial Protease-Like Activity Factor,CPAF),致病中發(fā)揮重要作用。干擾素基因刺激因子(stimulator of interferon genes,STING)和髓樣分化因子88(myeloiddifferentiationfactor88,MyD88)都是胞內(nèi)天然免疫反應(yīng)信號通路中的重要接頭蛋白。兩者借助共同下游信號通路與衣原體感染相關(guān),可能在其致病過程中發(fā)揮重要作用。鼠衣原體(Chiamydia muridarum,mouse pneumonitis agent,MoPn)基因結(jié)構(gòu)和組成與人型Ct極為相似。因此,用MoPn建模研究Ct致病機(jī)制。目的:本研究體外驗(yàn)證CPAF可能協(xié)助MoPn逃避胞外免疫監(jiān)視,進(jìn)而選STING-/-、MyD88-/-和C57BL/6J小鼠為對象,分別通過不同的MoPn感染途徑構(gòu)建動(dòng)物模型,檢測生殖道不同組織段的MoPn播散量、陰道清除速度、病理變化,分析STING、MyD88在生殖道功能屏障中的作用,研究衣原體可能的致病機(jī)制。方法:(1)用T細(xì)胞、DC及OT1或OT2抗原模擬抗原遞呈過程,與不同濃度CPAF作用,ELISA分別檢測上清液中IL-2的量。電泳分離及卡馬斯亮藍(lán)法,分析CPAF處理及未處理衣原體抗原組電泳條帶變化。分別選一種能被CPAF酶切及未能酶切的衣原體抗原代替OT2及OT1,重復(fù)抗原遞呈過程。ELISA分別檢測上清液IL-2的量。(2)用2X105IFUs MoPn經(jīng)不同途徑(陰道、宮腔、子宮遠(yuǎn)端段及卵巢囊)感染野生型、STING-/-、MyD88-/-小鼠。不同時(shí)間點(diǎn)處死小鼠,一部分生殖道大體評分、HE染色、組織和細(xì)胞免疫熒光技術(shù)(immunofluorescence,IF),對比組織病變程度。(3)一部分小鼠生殖道分段勻漿,IF檢測子宮-輸卵管屏障(uterotubal junction,U-T屏障)及宮頸屏障兩側(cè)衣原體量的差別。結(jié)果:1.CPAF選擇性抑制MHCⅡ-OT2抗原遞呈過程而非MHCⅠ-OT1(***P0.001)。CPAF同樣具有選擇性酶切衣原體T細(xì)胞抗原(***p0.001)。2.經(jīng)宮頸感染,STING-/-小鼠上生殖道大體病變和顯微鏡下炎癥評分比野生型更嚴(yán)重(**p0.01)。提示,STING可能參與U-T屏障功能。3.經(jīng)宮頸感染,MyD88-/-小鼠帶菌時(shí)間明顯延長(**P0.01)。經(jīng)宮頸及陰道感染均顯示,MyD88-/-小鼠上生殖道組織大體和微觀病變更嚴(yán)重(**P0.01)。提示,宮頸屏障及U-T屏障功能都可能因MyD88缺失而受影響。4.經(jīng)卵巢囊及子宮遠(yuǎn)端段感染,C57BL/6J小鼠U-T屏障兩側(cè)衣原體量有顯著差異(*p0.05),STING-/-及MyD88-/-差異不顯著(p0.05)。提示兩者參與U-T結(jié)構(gòu)屏障功能。結(jié)論:1)CPAF選擇性酶切Ct抗原,協(xié)助Ct逃避宿主胞外免疫監(jiān)視。2)STING信號通路主要通過參與U-T屏障功能,控制衣原體上行感染輸卵管。3)MyD88信號通路同時(shí)參與U-T屏障和宮頸屏障功能,抑制衣原體播散,控制衣原體上行感染輸卵管。
[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，

本文編號：1667385