定植因子CFA/I介导的肠道免疫对条件致病菌去定植的机制研究

The pathological basis of ICU acquired infection is the colonization of exogenous conditional pathogen in the intestine, moreover there is no ideal way to decolonize the exogenous conditional pathogen in the intestine. It was observed that the exogenous conditional pathogen could be decolonized by colonization factor antigen I (CFA/I) in the intestine, however the mechanisms is still unknown.The effects of CFA/I on the adhesion receptor in intestinal epithelial cells, the distribution and colonization of exogenous pathogen in gut, the activation of CFA/I on the intestinal humoral immune system and the effect of CFA/I on inflammatory reaction in the mucus layer, mucosa and lamina propria will be detected using laser scanning confocal,gene silencing and gene knockout methods. CD4+T subsets(Th1、Th2、Th17、Treg) will be separated from intestinal lamina propria of germ-free mice by Flow cytometry to investigate the effects of CFA/I on differentiation and maturation of CD4+ T cell subsets. The binding receptor of CFA/I on CD4+ T cells will be analyzed by silencing the TLR1 to TLR9 gene. Furthermore, the MyD88 gene or TRIF gene in Th1 and Th17 cells will be silenced to confirm the inflammatory signal pathway of CFA/I. To explore if the protective effects of CFA/I on gut barrier and intestinal microecological environment is associated with inflammatory reaction modulated by CFA/I through Th2 and Treg. The project provides a new way to prevent the ICU acquired infection.

肠道外源性致病菌定植是ICU获得性感染的病理基础，目前尚无理想方法对肠道致病菌进行去定植。作者研究显示定植因子CFA/I能促进肠道对外源性致病菌去定植，但其机制尚未阐明。本项目采用激光共聚焦、基因沉默和敲除等方法研究CFA/I对肠黏膜黏附分子受体的阻断作用，对致病菌在肠道内分布和定植的影响，对肠道体液免疫激活以及黏液层、肠黏膜和固有层内免疫应答反应的作用。应用流式细胞仪分离无菌小鼠肠黏膜固有层内CD4+T细胞亚群(Th1、Th2、Th17、Treg)，观察CFA/I对T细胞分化与成熟的影响；通过依次沉默Toll样受体1-9研究CFA/I作用的靶点；沉默Th1、Th17的MyD88和TRIF，明确CFA/I激活体内炎症反应信号通路；探讨CFA/I对肠黏膜屏障和肠道微生态环境保护作用是否与CFA/I通过Th2、Treg调控炎症反应有关。为降低ICU获得性感染开拓新途径。

肠道外源性致病菌定植是ICU获得性感染的病理基础，目前尚无理想方法对肠道致病菌进行去定植。本项目主要研究了定植因子CfaE（CFA/I尖端黏附素）对脓毒症小鼠肠道外源性致病菌定植的影响及其对脓毒症小鼠肠道外源性致病菌去定植的机制，并对LPS脓毒症模型下肠道菌群和固有免疫变化特征及相关性进行了探究。. 本项目首先探究CfaE对脓毒症小鼠肠道外源性致病菌定植的影响。采用电转法成功将pGFPuv 质粒转入大肠杆菌（H10407），使其表达绿色荧光。随后构建原核表达载体pET30a-CfaE-BL21，诱导表达并纯化了CfaE。采用盲肠穿孔结扎术构建脓毒症模型，对C57小鼠分成假手术组、假手术+环孢素A组、脓毒症组、脓毒症+CfaE 组和脓毒症+CfaE+环孢素A 组，后2组术后2h及第6d管饲CfaE，其余各组予生理盐水；各组小鼠于第6d给CfaE后2h管饲1×10^8CFU GFP-ETEC；于给GFP-ETEC后第1d、3d、7天、14d取小鼠新鲜粪便平板培养法选择培养GFP-ETEC及肠杆菌。研究发现：定植因子CfaE对脓毒症小鼠肠道外源性致病菌存在去定植作用且对肠道原籍肠杆菌有微弱保护作用，但对原籍条件致病菌无明显影响，同时可减轻脓毒症小鼠的体重丢失。. 为探究CfaE对脓毒症小鼠肠道外源性致病菌去定植的机制，我们对给或不给CfaE处理的脓毒症小鼠以及假手术组小鼠采集了肠道组织，应用RT-qPCR检测了肠道IL-22、RegIIIγ及RegIIIβ，分离了肠道免疫细胞，流式分析了IL-22+ILC3数量的变化，发现CfaE可上调脓毒症小鼠肠道IL-22、RegIIIβ、RegIIIγ的表达，并促进小肠ILC3s分泌IL-22。将CfaE和肠道免疫细胞体外共培养后检测IL-22+ILC3数量的变化，进一步证实CfaE可促进ILC3分泌IL-22。提示CfaE可能成为防治ICU获得性感染新途径。. 采用16SrRNA测序及流式细胞术对脓毒症小鼠发病后第0、2、7天肠道菌群及肠道固有免疫细胞的变化及两者的相关性进行分析，发现脓毒症导致肠道菌群及固有免疫发生了显著变化，菌群变化主要和ILC3s、MNPs数量和功能变化显著相关,特别是DCs亚群，ILC2s的变化及IMs分泌的IL-10、RMs分泌的TNF-α和肠道菌群的变化无显著相关性。