Enterococcus faecium EF1 调节新生仔猪空肠黏膜稳态的TLRs信号通路

Probiotic strain Enterococcus faecium EF1, isolated from the jejunal mucosa of a healthy piglet, has been shown to be effective in modulating cytokines expression and secretion in intestinal mucosa, as well as protecting intestinal mucosal barrier; however, the precise mechanism of action is not yet clear. Intestinal mucosa contains numerous epithelial cells and dendritic cells(DCs), which always work together and have collectively emerged as key players in the maintenance of intestinal homeostasis and immune defense. They can recognize a wide variety of microbial compounds using pattern receptor system which includes toll-like-receptor(TLR) and then elicit different immune responses. Therefore, this research is conducted to isolate DC cells from the jejunal mucosa lamina propria of healthy duroc × landrace × Yorkshire newborn piglets and Luchuan newborn piglets for investigating the pattern of the tight junction expression and cytokine production in the presence of the polarized porcine intestinal epithelial cells(IPEC-J2) and a polarized IPEC J2-DC Transwell coculture system actived by Enterococcus faecium EF1 and its LTA and DNA. Furthermore, the signaling pathways that are engaged by Enterococcus faecium EF1 and its components will be unveiled by gene silencing of tlr2/4/9, using specific siRNA. The result will clarify the interactions between Enterococcus faecium EF1 and the intestinal mucosa and it might pave the way for novel management strategies of livestock health and production.

Enterococcus faecium EF1来自健康仔猪空肠黏膜，前期试验研究表明它能影响仔猪肠黏膜表达和分泌细胞因子，保护肠黏膜屏障，但机理尚不清楚。肠上皮细胞和肠黏膜树突细胞（DC）是维持肠黏膜先天免疫系统稳态的重要免疫细胞。它们协同作用，并通过表达Toll-样受体（TLR）识别微生物配体，产生先天免疫应答。本课题建立极化的IPEC-J2细胞模型，并从杜长大新生仔猪和广西陆川猪新生仔猪空肠黏膜中分离培养DC细胞，分别与IPEC-J2细胞建立起 (IPEC J2-DC)细胞共培养体系，研究Enterococcus faecium EF1对细胞培养体系的紧密连接蛋白表达和细胞因子分泌的影响，并通过tlr基因沉默方法研究其TLRs信号通路，探明Enterococcus faecium EF1维持仔猪肠黏膜稳态的作用机理，为该益生菌株在猪的健康养殖生产中推广应用提供科学依据。

为了探明益生屎肠球菌EF1调节仔猪小肠粘膜免疫稳态的作用机理，制备了屎肠球菌的脂磷壁酸（LTA）；用屎肠球菌的活菌和热灭活菌对哺乳期仔猪进行试验；在IPEC-J2培养14天内建立了极化模型，研究活菌、热灭活菌及其细胞壁对极化的IPEC-J2的影响；分离猪外周血树突细胞并建立IPEC J2-树突细胞共培养体系。结果表明，1）对数生长期屎肠球菌的LTA粗提物得率约为1%，经疏水作用和离子交换层析后的LTA得率为0.03%-0.044%。2）屎肠球菌（活菌和灭活菌）主要是促进了仔猪小肠黏膜和IPEC-J2的occludin和claudin或zo-1蛋白的表达。3）屎肠球菌对7日龄仔猪小肠黏膜多种细胞因子应答的免疫刺激活性都很强，其中TGF-β的基因表达量或分泌量普遍提高，但是21日龄仔猪小肠黏膜的免疫反应明显减弱，只诱导IL-8或TNF-α表达或分泌；此外也促进IPEC-J2细胞表达和分泌TGF-β，并引起il-8基因表达上调，却抑制IL-8和IL-10的分泌。4）屎肠球菌活菌对7日龄仔猪空肠黏膜以及猪空肠上皮细胞IPEC-J2的tlr-2和tlr-4基因表达都有诱导作用，而且Toll-样受体的应答与细胞因子应答比较吻合。5）活菌还直接或间接地影响仔猪结肠微生物群落不同的优势门或优势属；活菌和热灭活菌都能够促进某些小肠消化酶的分泌，并且对仔猪腹泻率的减少有相似的作用。结果提示，屎肠球菌（活菌和热灭活菌）对仔猪小肠黏膜结构屏障完整性都有一定的保护作用，活菌可能经由TLR-2和TLR-4信号传导途径而诱导空肠黏膜促炎或抗炎细胞因子的应答，或者还通过调节消化生理机能和肠道微生态平衡来维持仔猪肠道稳态。