肠道相关树突状细胞参与创伤性休克肠粘膜屏障功能改变的研究

发布时间：2018-04-19 14:26

  本文选题：固有层树突状细胞 + 肠系膜淋巴结　； 参考：《浙江大学》2015年博士论文

【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
[Abstract]:Background :

Severe trauma shock is often caused by the inhibition of the immune function of the organism , which leads to severe systemic infection , sepsis ( sepsis ) and multiple organ failure ( MOF ) . Our previous studies have found that severe trauma can alter the apoptosis and differentiation balance ( Th2 response ) of CD4 + T lymphocytes , which in turn affects the immune status of the whole body , yet the exact mechanism is not yet clear .

In recent years , the study has focused on gut - related dendritic cells and intestinal epithelial cells , which have been focused on gut - related dendritic cells and intestinal epithelial cells . They have been focused on gut - related dendritic cells and intestinal epithelial cells .

The dendritic cells of the mesenteric lymph nodes and the lamina propria of the intestinal mucosa are two main gut - related dendritic cells , which act as antigen - presenting cells to " direct " the local immune response of the intestinal tract . In particular , the LPDCs , which are the first immune barrier in the small intestine mucosa , have been questioned . In particular , the cells of the lamina propria of CD11chiCD11bhi have been defined as small intestine LPDCs for the first time .

On the other hand , it has been found that there are 1012 bacteria and several lethal doses of endotoxin in the normal intestine . However , intestinal bacteria and endotoxin have failed to enter the circulation , but in addition to the common defense of the mechanical and immune barriers , it is possible to prevent direct contact of pathogenic bacteria with the intestinal epithelium , thereby maximizing the role of the " biological barrier " .

Therefore , we have speculated that severe traumatic shock , on the one hand , changes the function of gut - related dendritic cells through the mechanisms of ischemia - reperfusion injury and so on , which leads to the decrease of intestinal immunity and the disorder of immune status of the whole body ;
On the other hand , the intestinal mucosal epithelium is directly damaged by a large number of bacteria caused by intestinal pathogenic bacteria , which further increases the damage of the intestinal barrier . However , the hypothesis of the above - mentioned mechanism still needs the exact evidence .

Purpose :

In this study , the gut - related dendritic cells and intestinal epithelium were used as the key cells of intestinal immune barrier . The function of dendritic cells and intestinal mucosal epithelial injury were directly affected by traumatic shock .

Method :

1 ) Based on " long bone fracture + hemorrhagic shock " , a stable traumatic shock rat model was established by mean arterial pressure of 30 mmHg as the shock standard , and the degree of maturation and apoptosis after traumatic shock was studied by mesenteric lymph node dendritic cells ( MLN - DCs ) , and the changes of the differentiation ability of naive CD4 + T cells to the various CD4 + T helper cells ( Treg , Th1 , Th2 ) were studied .

2 ) On the basis of establishing the mouse model of wound - shock mice and the cultivation of T _ 1 - 5 - / - mice , the specific expression of Toll - like receptor - 5 ( T1r5 ) and specific non - T cell - dependent immune activation ( retinol ( RA ) - mediated immune activation ( retinol ( RA ) - mediated ) signaling pathway were used as the key cells . Through the differentiation of naive CD4 + T cells to Th1 and Th17 , the secretion of retinol dehydrogenase ( RALDH ) and the change of intestinal bacterial translocation after trauma shock were detected .

3 ) We have obtained several enterogenous vancomycin - resistant enterococcus faecium ( VRE ) in clinic , and have confirmed that it has stronger intestinal epithelial damage characteristics , which are respectively planted on the caco2 model , and electron microscope observation , caco2 cell layer voltage detection and the detection of cytokines such as TNF - 伪 are used to confirm the mechanism of direct injury of intestinal epithelial membrane barrier by intestinal pathogenic bacteria .

Results :

1)澶ч紶MLN-DCs鍦ㄥ垱浼ゆ，

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