【作者】 张匀；

【导师】 梁廷波；

【作者基本信息】 浙江大学 ， 临床医学， 2015， 博士

【摘要】 背景：严重创伤休克常因为导致机体免疫功能抑制,进而诱发全身严重感染、脓毒血症(sepsis)及多脏器功能衰竭(MOF)。我们的前期研究已经发现严重创伤可以改变CD4+T淋巴细胞的凋亡及分化平衡(Th2反应)进而影响全身的免疫状态,然而确切的机制尚未明确。小肠作为一个极易受到缺血-再灌注损伤的器官、最大的免疫器官,同时又接触最大量的肠腔内致病菌,一直以来被认为是危重症患者并发院内感染及MODS的“发动机”。近年来的研究已经聚焦在肠道相关树突状细胞及肠上皮细胞上,它们在接受缺血再灌注损伤及肠道致病菌的双重攻击下可通过诱导淋巴细胞的分化异常进而导致肠道局部及全身的免疫状态处于紊乱状态。严重创伤休克打击下它们将如何工作可能会成为机体遭受严重感染的关键机制。肠系膜淋巴结树突状细胞及肠粘膜固有层树突状细胞为两组主要的肠道相关树突状细胞,它们作为抗原递呈细胞“指挥”着肠道局部的免疫反应。尤其是作为小肠黏膜第一道免疫屏障的LPDCs一直少有人问津,Satoshi Uematsu首次定义CD11chiCD11bhi的固有层细胞为小肠LPDCs,同时发现其分别通过独特的方式实现自身活化、对T、B细胞分化及淋巴细胞“回巢”的诱导,这些重要的发现可能成为创伤／肠粘膜免疫研究新的趋势。另一方面,现已发现正常肠内即有1012个细菌和数倍致死量的内毒素。但肠道细菌和内毒素未能进入到循环中,除了由于机械与免疫屏障的共同防御外,还由于100-1000倍于需氧菌的厌氧菌占据邻近上皮细胞间空隙,最大可能地防止致病菌与肠上皮的直接接触,从而发挥了“生物屏障”的作用。但当小肠遭遇I/R损伤尤其是临床治疗使用大量光谱抗生素之后,这些正常保护机制被破坏,最终导致肠道发生严重的炎症反应及腹泻,同时大量条件性肠道致病菌及其毒素的移位促成全身的严重感染。由此我们推测：严重创伤休克一方面通过缺血再灌注损伤等机制改变肠道相关树突状细胞的功能进而导致肠道免疫防御能力的降低以及全身的免疫状态的紊乱；另一方面由于肠道致病菌的大量滋生直接损伤肠粘膜上皮,进而加重肠道屏障的损伤。但上述机制的假说尚需确切的证据。目的：本研究将以肠道相关树突状细胞以及肠上皮作为肠道免疫屏障的关键细胞,以创伤休克直接影响树突状细胞的功能以及肠道致病菌致肠粘膜上皮损伤作为两个研究思路,深入研究创伤休克后肠道免疫屏障损伤、细菌移位发生及机体遭受严重感染的重要参与机制。方法：1)以“长骨骨折+失血休克”为基本内容,以平均动脉压30mmHg作为休克标准建立稳定的创伤性休克大鼠模型,以肠系膜淋巴结树突状细胞(MLN-DCs)作为对象,研究其在创伤性休克打击后的成熟程度、凋亡变化的情况,以及诱导naive CD4+T细胞向各CD4+T辅助细胞各亚群(Treg,Th1, Th2)分化能力的改变。2)在建立创伤性休克小鼠模型及Tlr5-/-小鼠培育的基础上,以特异性表达Toll样受体-5(Tlr5)和特有的非T细胞依赖免疫激活(视黄醇(RA)介导)信号途径的肠粘膜下固有层树突状细胞(LPDCs)为关键细胞,通过对其诱导naive CD4+T细胞向Th1及Th17分化、合成分泌视黄醇脱氢酶(RALDH)以及以生物荧光标记的柠檬酸杆菌作为标记检测创伤休克后肠道细菌移位的改变的检测。3)我们在临床获得数株肠源性耐万古霉素屎肠球菌(VRE),并已证实其具有较强的致肠上皮损伤的特性,分别种植于caco2模型上,采用电镜观察、caco2细胞层电压检测以及TNF-α等细胞因子的检测等方法以证实临床验证的肠道致病菌直接损伤肠上皮面膜屏障的机制。结果：1)大鼠MLN-DCs在创伤性休克发生后的早期即出现成熟度(CD80,CD86,MHCⅡ)下降、凋亡增加,并且诱导naive CD4+T细胞向Th2及Treg细胞分化的趋势。2)创伤性休克可以导致小肠LPDCs合成RA的能力出现明显下降,由此使得LPDCs诱导Thl及Th17分化下调。正常情况下Tlr5-KO小鼠小肠LPDCs合成RA及诱导Th1分化的能力较野生型明显减弱,但创伤休克发生后变化不明显。创伤性休克可以导致野生型小鼠小肠内细菌移位明显增加,但并未导致Tlr5-KO小鼠肠道细菌向血液及肝脏的移位明显增加3)相比较于未致肠炎的肠球菌,临床获得的确证引起危重患者合并细菌性肠炎的VRE作为一种肠道致病菌能够导致caco2细胞的病理损伤及细胞层电压(TEER)的下降,同时合成大量炎症介质。结论：严重创伤性休克不仅能够通过抑制肠道相关树突状细胞的功能以削弱肠道黏膜免疫屏障的功能及机体免疫稳态,同时通过诱导肠道致病菌直接损伤肠上皮,进而导致肠道致病菌大量肠外移位及全身严重感染的发生更多还原

【Abstract】 BackgroundIt’s well known that Trauma/hemorrhagic shock (TH/S) is associated with immunosuppression, which can render patients susceptible to nosocomial infection and multiorgan dysfunction. We have previously demonstrated in a rat model of TH/S the typical differentiation towards Th2phenotype in the T-cell population with an associated increase in the apoptosis of CD4+T-cells. However, the mechanisms leading to TH/S immunosuppression are poorly understood.As one of the most vulnerable organs under ischemia reperfusion injury, an organ access to the largest amount of bacteria and one of the largest immune organ, the gut has been hypothesized to be the "motor" of nosocomial infection and multiple organ dysfunction syndrome (MODS). Under the attacking of ischemia reperfusion and pathogen, the destroyed mucosal immune system (including dendritic cells and epithelium) will induce local and systemic immunosuppression consequent with bacterial translocation.MLN-DCs and LPDCs as the main part of gut associated DCs are thought to be critical in "decision" of whether to tolerant or protective immune response in the intestinal immune barrier. A subset of CD11chiCD11bhi lamina propria dendritic cells (LPDCs) discovered by Satoshi Uematsu has been identified that expressed Toll-like receptor5(TLR5) in the small intestine. When stimulated by the TLR5ligand flagellin, TLR5+LPDCs can induce the differentiation of naive B cells into immunoglobulin A-producing plasma cells and of antigen-specific interleukin17-producing T helper cells and type1T helper cells. More interesting, unlike spleen DCs, the LPDCs specifically produced retinoic acid (RA), which is regulated by Tlr5-Myd88signaling system and in a dose-dependent way regulated the differentiation interleukin17-producing T helper cells in the lamina propria.The adult human intestine is home to more than100trillion bacteria, which dwarves the number of somatic and germ cells in the entire human organism. There are estimated to be between500-1000species of bacteria present in our intestines. Aerobic, facultative, and anaerobic bacteria are all part of the enteric microflora. It was found that broad-spectrum antibiotics were able to promote the development of pathogenic bacteria, which will finally induce infectious diarrhea and extraenteric infections.We make a hypothesis that weakness of intestinal immune barrier and disorder of immune state of the body induced by TH/S companied with the large number of intestinal pathogenic bacteria growth result in the increase the bacterial translocation and severe infection, and the mechanism of the hypothesis needs conclusive evidence. Aim:With the gut associated dendritic cells and intestinal epithelial cells as the key cells of intestinal immune barrier, the disfunction of dendritic cells and intestinal mucosal damage induced by intestinal bacteria as the two research thinking, this study will try find out the important mechanism of the injury of intestinal immune barrier and bacterial translocation after traumatic shock. Methods:1) Evaluate the effects of TH/S on the function of MLN-DCs in a Sprague-Dawley rat model with mid-diaphyseal transverse fracture and an MAP of3±5mmHg for90min. MLN-DCs were evaluated for surface expression of CD80, CD86, and MHC II, as well as for apoptosis. The in-vitro and in-vivo effects of MLN-DCs-induced induction of CD4T cell differentiation, and the underlying mechanisms were investigated.2) Under the help of TH/S mice model and TLR5-/-KO mice, this study used the LPDCs as the key cells with the characteristic of Toll-like5receptor and retinoic acid (RA) production, which is regulated by T1r5-Myd88signaling system and in a dose-dependent way regulated the differentiation of thl and th17cells. Eexamined the effects of TH/S on the function of LPDCs on induction of CD4T cell differentiation especially TH1and TH17, and the underlying mechanisms such as the RALDH synthetic ability. Additionally the intra-intestinal bacterial translocation was monitored using bioluminescent citrobacter and IVIS Spectrum optical imaging system.3) Several VRE samples which induced severe enteritis and extraenteric disseminations were used to infect the human intestinal Caco-2cell line, followed by inflammatory cytokine assays and morphological characterization by electron microscopy.Results:1) T/HS-induced increase in early apoptosis of MLN-DCs which was likely mediated by cleaved-caspase-3and-9, AKT and ERK. CD80and MHC-II expression levels were downregulated in the T/HS group compared to the SHAM, Importantly, MLN-DCs from T/HS rats promoted differentiation of CD4+T-cells to Th2and Treg cells. The partial amelioration of these trends by allicin treatment could be mediated by STAT1and STAT6.2) LPDCs from wide type TH/S mice induced lower differentiation ratio of CD4+T cells towards Thl and Thl7especially under severe TH/S attack, in TLR5KO mice with or without TH/S the ability of LPDCs of inducing differentiation ratio of CD4+T cells towards Thl and Th17was low, which likely mediated by the ability of RA production. In wild type mice, TH/S can induce a higher ratio of citrobacter translocation to liver and blood. To our interesting, there is low ratio of citrobacter translocation in TLR5KO mice with or without TH/S.3) All six VRE strains isolated from stool samples caused severe enteritis. The same strains further inflicted significant damage and induced inflammatory reactions in Caco-2cells. Homologous assays demonstrated high homology between samples from stool and peripancreatic effusions in two patients, indicating the occurrence of extraenteric disseminations.Conclusion: T/HS resulted in less functionally mature gut associated DCs and significant DCsrelated immunosuppression, and the gut sourced pathogenic bacteria may causeinflammatory injuries to enteric epithelium during the course of TH/S, which resultingin enteritis and extraenteric disseminations.更多还原