本文選題：固有層樹突狀細胞 + 腸系膜淋巴結�。� 參考：《浙江大學》2015年博士論文

【摘要】：背景： 嚴重創(chuàng)傷休克常因為導致機體免疫功能抑制,進而誘發(fā)全身嚴重感染、膿毒血癥(sepsis)及多臟器功能衰竭(MOF)。我們的前期研究已經發(fā)現嚴重創(chuàng)傷可以改變CD4+T淋巴細胞的凋亡及分化平衡(Th2反應)進而影響全身的免疫狀態(tài),然而確切的機制尚未明確。 小腸作為一個極易受到缺血-再灌注損傷的器官、最大的免疫器官,同時又接觸最大量的腸腔內致病菌,一直以來被認為是危重癥患者并發(fā)院內感染及MODS的“發(fā)動機”。近年來的研究已經聚焦在腸道相關樹突狀細胞及腸上皮細胞上,它們在接受缺血再灌注損傷及腸道致病菌的雙重攻擊下可通過誘導淋巴細胞的分化異常進而導致腸道局部及全身的免疫狀態(tài)處于紊亂狀態(tài)。嚴重創(chuàng)傷休克打擊下它們將如何工作可能會成為機體遭受嚴重感染的關鍵機制。 腸系膜淋巴結樹突狀細胞及腸粘膜固有層樹突狀細胞為兩組主要的腸道相關樹突狀細胞,它們作為抗原遞呈細胞“指揮”著腸道局部的免疫反應。尤其是作為小腸黏膜第一道免疫屏障的LPDCs一直少有人問津,Satoshi Uematsu首次定義CD11chiCD11bhi的固有層細胞為小腸LPDCs,同時發(fā)現其分別通過獨特的方式實現自身活化、對T、B細胞分化及淋巴細胞“回巢”的誘導,這些重要的發(fā)現可能成為創(chuàng)傷／腸粘膜免疫研究新的趨勢。 另一方面,現已發(fā)現正常腸內即有1012個細菌和數倍致死量的內毒素。但腸道細菌和內毒素未能進入到循環(huán)中,除了由于機械與免疫屏障的共同防御外,還由于100-1000倍于需氧菌的厭氧菌占據鄰近上皮細胞間空隙,最大可能地防止致病菌與腸上皮的直接接觸,從而發(fā)揮了“生物屏障”的作用。但當小腸遭遇I/R損傷尤其是臨床治療使用大量光譜抗生素之后,這些正常保護機制被破壞,最終導致腸道發(fā)生嚴重的炎癥反應及腹瀉,同時大量條件性腸道致病菌及其毒素的移位促成全身的嚴重感染。 由此我們推測：嚴重創(chuàng)傷休克一方面通過缺血再灌注損傷等機制改變腸道相關樹突狀細胞的功能進而導致腸道免疫防御能力的降低以及全身的免疫狀態(tài)的紊亂；另一方面由于腸道致病菌的大量滋生直接損傷腸粘膜上皮,進而加重腸道屏障的損傷。但上述機制的假說尚需確切的證據。 目的： 本研究將以腸道相關樹突狀細胞以及腸上皮作為腸道免疫屏障的關鍵細胞,以創(chuàng)傷休克直接影響樹突狀細胞的功能以及腸道致病菌致腸粘膜上皮損傷作為兩個研究思路,深入研究創(chuàng)傷休克后腸道免疫屏障損傷、細菌移位發(fā)生及機體遭受嚴重感染的重要參與機制。 方法： 1)以“長骨骨折+失血休克”為基本內容,以平均動脈壓30mmHg作為休克標準建立穩(wěn)定的創(chuàng)傷性休克大鼠模型,以腸系膜淋巴結樹突狀細胞(MLN-DCs)作為對象,研究其在創(chuàng)傷性休克打擊后的成熟程度、凋亡變化的情況,以及誘導naive CD4+T細胞向各CD4+T輔助細胞各亞群(Treg,Th1, Th2)分化能力的改變。 2)在建立創(chuàng)傷性休克小鼠模型及Tlr5-/-小鼠培育的基礎上,以特異性表達Toll樣受體-5(Tlr5)和特有的非T細胞依賴免疫激活(視黃醇(RA)介導)信號途徑的腸粘膜下固有層樹突狀細胞(LPDCs)為關鍵細胞,通過對其誘導naive CD4+T細胞向Th1及Th17分化、合成分泌視黃醇脫氫酶(RALDH)以及以生物熒光標記的檸檬酸桿菌作為標記檢測創(chuàng)傷休克后腸道細菌移位的改變的檢測。 3)我們在臨床獲得數株腸源性耐萬古霉素屎腸球菌(VRE),并已證實其具有較強的致腸上皮損傷的特性,分別種植于caco2模型上,采用電鏡觀察、caco2細胞層電壓檢測以及TNF-α等細胞因子的檢測等方法以證實臨床驗證的腸道致病菌直接損傷腸上皮面膜屏障的機制。 結果： 1)大鼠MLN-DCs在創(chuàng)傷性休克發(fā)生后的早期即出現成熟度(CD80,CD86,MHCⅡ)下降、凋亡增加,并且誘導naive CD4+T細胞向Th2及Treg細胞分化的趨勢。 2)創(chuàng)傷性休克可以導致小腸LPDCs合成RA的能力出現明顯下降,由此使得LPDCs誘導Thl及Th17分化下調。正常情況下Tlr5-KO小鼠小腸LPDCs合成RA及誘導Th1分化的能力較野生型明顯減弱,但創(chuàng)傷休克發(fā)生后變化不明顯。創(chuàng)傷性休克可以導致野生型小鼠小腸內細菌移位明顯增加,但并未導致Tlr5-KO小鼠腸道細菌向血液及肝臟的移位明顯增加 3)相比較于未致腸炎的腸球菌,臨床獲得的確證引起危重患者合并細菌性腸炎的VRE作為一種腸道致病菌能夠導致caco2細胞的病理損傷及細胞層電壓(TEER)的下降,同時合成大量炎癥介質。 結論： 嚴重創(chuàng)傷性休克不僅能夠通過抑制腸道相關樹突狀細胞的功能以削弱腸道黏膜免疫屏障的功能及機體免疫穩(wěn)態(tài),同時通過誘導腸道致病菌直接損傷腸上皮,進而導致腸道致病菌大量腸外移位及全身嚴重感染的發(fā)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 - 偽 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，

本文編號：1773467