Myd88在肠源性VRE感染致小肠粘膜上皮细胞免疫功能改变的机制研究

Dysbacteriosis occured in critical illness's bowel plays an important role in leading to sepsis and MOF. Addition to as a barrier, the epithelium also has the immunal functions, which as a whole proceed to protect against invasion of potentially harmful antigens. We have obtained several strains of enterogenous Vancomycin-resistant Enterococcus faecium (VRE) in bowel of 4 critical illness patients, which have been proved to have strong ability of inducing intestinal epithelial injury, and the gene of potential signaling pathway molecules (Myd88 pathway) which take part in intestinal epithelial damage were also found abnormal expressed. But the exact mechanism of the specific immune response and defense response occurred in the intestinal epithelium has not been elucidated. This project intends to illustrate the ways and the mechanisms take part in the changes of the immunity reaction of intestinal epithelial induced by VRE with the use of experimental platform in vitro (caco2 model) and in vivo (part of the bowel infection model in mouse) and the feather use of intestinal epithelial Myd88 gene knockout mice. This work will not only be able to provide an explanation for how the flora lead to the spread of infection, and are more likely to provide a theoretical basis for new treatment which can enhance the ability of pathogenic defense of intestinal epithelial.

危重症患者菌群失调是导致条件致病菌发生移位和脓毒血症（SEPSIS）、多脏器功能衰竭(MOF)发生的重要因素。小肠上皮不仅构筑机械屏障,同时又具有免疫活性,在抵御病原菌的侵袭中起关键作用。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,并初步发现受损肠上皮内信号通路分子相关基因（Myd88通路）有异常表达，但尚未阐明肠上皮对其产生特异性免疫应答及防御反应的确切机理。本项目拟分别采用体外（caco2模型）及体内（小鼠部分肠段感染模型）的实验平台，并利用已具备的肠上皮Myd88基因敲除小鼠等技术进行深入研究，以达到阐明上述肠源性VRE感染致小肠粘膜上皮细胞免疫功能改变的方式及机制，并深入探讨Myd88信号通路在其中的作用。此项工作不但能够为菌群失调如何导致感染扩散提供解释，更有可能为今后开发以提升肠上皮防御致病菌能力为靶标的新型治疗提供理论依据。

危重症患者菌群失调是导致条件致病菌发生移位和脓毒血症（SEPSIS）、多脏器功能衰竭(MOF) 发生的重要因素。小肠上皮不仅构筑机械屏障, 同时又具有免疫活性, 在抵御病原菌的侵袭中起关键作用。前期，我们在临床获得数株肠源性耐万古霉素屎肠球菌（VRE）,通过感染caco2细胞单层模型证实了其具有较强的致肠上皮损伤的特性, 并通过细菌同源性分析（包括药敏试验（Minimum Inhibitory Concentration, MIC法）、多位点测序分型（Multilocus sequence typing, MLST）、脉冲场电泳（Pulsed-field gel electrophoresis，PFGE））证实了部分患者出现了肠道细菌移位，且通过Microarray初步发现了受损肠上皮内信号通路分子相关基因（Myd88通路）有异常表达，但尚未阐明肠上皮对其产生特异性免疫应答及防御反应的确切机理。所以，为了阐明上述肠源性VRE 感染致小肠粘膜上皮细胞免疫功能改变的方式及机制，并深入探讨Myd88信号通路在其中的作用，本项目分别采用了体外（caco2模型）及体内（小鼠部分肠段感染模型）的实验平台，并利用已具备的肠上皮 Myd88 基因敲除小鼠等技术进行深入研究。我们有了如下新的发现：1）致肠炎VRE感染caco2细胞单层模型后引起上清液IL-8、IL-6、TNF-α急剧升高，直接或间接诱导了促炎性T细胞免疫反应，而在感染菌量增加到一定程度后又出现了免疫抑制，能诱导细胞内RALDH的合成增加；致肠炎VRE感染小鼠肠道后，肠道粘膜层出现了明显的病理损伤，受感染肠上皮细胞（IEC）或LPDC均能诱导naïve T分化，趋势与体外实验一致，受致肠炎VRE感染后，小肠上皮细胞视黄醛脱氢酶（ RALDH）的表达明显上升；TLR5敲除小鼠来源的肠上皮细胞、肠上皮myd88条件性敲除小鼠来源的肠上皮表达RALDH均下降, 而CD11c-DC myd88条件性敲除小鼠来源的肠上皮细胞表达RALDH轻度上升。我们还利用利用生物性发光柠檬酸杆菌示踪肠道细菌移位，发现RA能够显著减少柠檬酸杆菌的移位及提高致肠炎VRE感染后小鼠的生存率。总体上来说，此项工作不但能够为菌群失调如何导致感染扩散提供解释，更有可能为今后开发以提升肠上皮防御致病菌能力为靶标的新型治疗提供理论依据。