本文選題：固有層樹突狀細(xì)胞 + 腸系膜淋巴結(jié)　； 參考：《浙江大學(xué)》2015年博士論文

【摘要】：背景： 嚴(yán)重創(chuàng)傷休克常因?yàn)閷?dǎo)致機(jī)體免疫功能抑制,進(jìn)而誘發(fā)全身嚴(yán)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(jīng)發(fā)現(xiàn)嚴(yán)重創(chuàng)傷可以改變CD4+T淋巴細(xì)胞的凋亡及分化平衡(Th2反應(yīng))進(jìn)而影響全身的免疫狀態(tài),然而確切的機(jī)制尚未明確。 小腸作為一個(gè)極易受到缺血-再灌注損傷的器官、最大的免疫器官,同時(shí)又接觸最大量的腸腔內(nèi)致病菌,一直以來被認(rèn)為是危重癥患者并發(fā)院內(nèi)感染及MODS的“發(fā)動機(jī)”。近年來的研究已經(jīng)聚焦在腸道相關(guān)樹突狀細(xì)胞及腸上皮細(xì)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(dǎo)淋巴細(xì)胞的分化異常進(jìn)而導(dǎo)致腸道局部及全身的免疫狀態(tài)處于紊亂狀態(tài)。嚴(yán)重創(chuàng)傷休克打擊下它們將如何工作可能會成為機(jī)體遭受嚴(yán)重感染的關(guān)鍵機(jī)制。 腸系膜淋巴結(jié)樹突狀細(xì)胞及腸粘膜固有層樹突狀細(xì)胞為兩組主要的腸道相關(guān)樹突狀細(xì)胞,它們作為抗原遞呈細(xì)胞“指揮”著腸道局部的免疫反應(yīng)。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(xì)胞為小腸LPDCs,同時(shí)發(fā)現(xiàn)其分別通過獨(dú)特的方式實(shí)現(xiàn)自身活化、對T、B細(xì)胞分化及淋巴細(xì)胞“回巢”的誘導(dǎo),這些重要的發(fā)現(xiàn)可能成為創(chuàng)傷／腸粘膜免疫研究新的趨勢。 另一方面,現(xiàn)已發(fā)現(xiàn)正常腸內(nèi)即有1012個(gè)細(xì)菌和數(shù)倍致死量的內(nèi)毒素。但腸道細(xì)菌和內(nèi)毒素未能進(jìn)入到循環(huán)中,除了由于機(jī)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(jù)鄰近上皮細(xì)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(dāng)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(hù)機(jī)制被破壞,最終導(dǎo)致腸道發(fā)生嚴(yán)重的炎癥反應(yīng)及腹瀉,同時(shí)大量條件性腸道致病菌及其毒素的移位促成全身的嚴(yán)重感染。 由此我們推測：嚴(yán)重創(chuàng)傷休克一方面通過缺血再灌注損傷等機(jī)制改變腸道相關(guān)樹突狀細(xì)胞的功能進(jìn)而導(dǎo)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(jìn)而加重腸道屏障的損傷。但上述機(jī)制的假說尚需確切的證據(jù)。 目的： 本研究將以腸道相關(guān)樹突狀細(xì)胞以及腸上皮作為腸道免疫屏障的關(guān)鍵細(xì)胞,以創(chuàng)傷休克直接影響樹突狀細(xì)胞的功能以及腸道致病菌致腸粘膜上皮損傷作為兩個(gè)研究思路,深入研究創(chuàng)傷休克后腸道免疫屏障損傷、細(xì)菌移位發(fā)生及機(jī)體遭受嚴(yán)重感染的重要參與機(jī)制。 方法： 1)以“長骨骨折+失血休克”為基本內(nèi)容,以平均動脈壓30mmHg作為休克標(biāo)準(zhǔn)建立穩(wěn)定的創(chuàng)傷性休克大鼠模型,以腸系膜淋巴結(jié)樹突狀細(xì)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(dǎo)naive CD4+T細(xì)胞向各CD4+T輔助細(xì)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(chǔ)上,以特異性表達(dá)Toll樣受體-5(Tlr5)和特有的非T細(xì)胞依賴免疫激活(視黃醇(RA)介導(dǎo))信號途徑的腸粘膜下固有層樹突狀細(xì)胞(LPDCs)為關(guān)鍵細(xì)胞,通過對其誘導(dǎo)naive CD4+T細(xì)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(biāo)記的檸檬酸桿菌作為標(biāo)記檢測創(chuàng)傷休克后腸道細(xì)菌移位的改變的檢測。 3)我們在臨床獲得數(shù)株腸源性耐萬古霉素屎腸球菌(VRE),并已證實(shí)其具有較強(qiáng)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(xì)胞層電壓檢測以及TNF-α等細(xì)胞因子的檢測等方法以證實(shí)臨床驗(yàn)證的腸道致病菌直接損傷腸上皮面膜屏障的機(jī)制。 結(jié)果： 1)大鼠MLN-DCs在創(chuàng)傷性休克發(fā)生后的早期即出現(xiàn)成熟度(CD80,CD86,MHCⅡ)下降、凋亡增加,并且誘導(dǎo)naive CD4+T細(xì)胞向Th2及Treg細(xì)胞分化的趨勢。 2)創(chuàng)傷性休克可以導(dǎo)致小腸LPDCs合成RA的能力出現(xiàn)明顯下降,由此使得LPDCs誘導(dǎo)Thl及Th17分化下調(diào)。正常情況下Tlr5-KO小鼠小腸LPDCs合成RA及誘導(dǎo)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(dǎo)致野生型小鼠小腸內(nèi)細(xì)菌移位明顯增加,但并未導(dǎo)致Tlr5-KO小鼠腸道細(xì)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(xì)菌性腸炎的VRE作為一種腸道致病菌能夠?qū)е耤aco2細(xì)胞的病理損傷及細(xì)胞層電壓(TEER)的下降,同時(shí)合成大量炎癥介質(zhì)。 結(jié)論： 嚴(yán)重創(chuàng)傷性休克不僅能夠通過抑制腸道相關(guān)樹突狀細(xì)胞的功能以削弱腸道黏膜免疫屏障的功能及機(jī)體免疫穩(wěn)態(tài),同時(shí)通過誘導(dǎo)腸道致病菌直接損傷腸上皮,進(jìn)而導(dǎo)致腸道致病菌大量腸外移位及全身嚴(yán)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