乳酸杆菌调控猪肠干细胞修复受损肠黏膜机制的研究

Mildew diet, stress and pathogen infection are the main cause of pig intestinal mucosal damage. However, the replacement of damaged intestinal epithelial cells by the proliferation and differentiation of intestinal stem cell is the major regulatory targets to repair the damaged mucosal structure. The most feature of intestinal stem cells is that intestinal flora could contact and modulate the intestinal stem cell directly, but the molecular regulation mechanism of Lactobacillus to intestinal stem cell is unclear. Therefore, this project will use the three-dimensional model porcine enteroids containing intestinal stem cells. Firstly, under the pathological injury of intestinal mucosal treated with Salmonella or dextran sodium sulfate (DSS), to study the regulation of Lactobacillus to the key target genes related to pattern recognition receptors (PRRs) and Reactive oxygen species (ROS) signaling pathway in intestinal stem cells, and confirm the function of the target genes to intestinal stem cells by interfering and over-expression; then in-depth study the mechanism of the cooperation of PRRs and ROS signaling pathways (including ROS concentration and Duox/Nox selectivity dependent pathway) controlled by the target genes, as well as to regulate Wnt and Notch signaling pathway of intestinal stem cell to repair the damaged intestinal mucosa structure. This project will reveal the regulation mechanism of Lactobacillus to the proliferation and differentiation of intestinal stem cells to repair damaged mucosa, guarantee efficient absorption of nutrients and mucosal immunity, as well as provide theoretical basis for the use of Lactobacillus to maintain intestinal barrier.

霉变饲料、应激和病原微生物感染是猪肠黏膜损伤的主要原因，而肠干细胞通过自身的增殖分化替代损伤的肠上皮细胞是修复受损肠黏膜结构的的主要调控靶点。肠干细胞的特点在于其游离面直接与肠道菌群接触并受其调控，但目前肠道菌群（如乳酸杆菌）调控肠干细胞修复受损肠黏膜的分子机制尚不清楚。因此本项目将建立含有肠干细胞的猪肠enteroids三维立体模型，研究沙门氏菌或DSS导致肠黏膜损伤下，乳酸杆菌对肠干细胞PRRs和ROS等信号通路关键靶基因的调控，并通过干扰和过表达证实靶基因的功能；深入研究靶基因控制的PRRs和ROS信号通路（包括ROS浓度和Duox/Nox选择性依赖途径），以及共同调控猪肠干细胞Wnt和Notch信号通路修复损伤肠黏膜结构的作用机制。本项目将揭示乳酸杆菌调控猪肠干细胞的增殖分化并修复受损肠黏膜结构的作用机制，保障营养物质吸收和黏膜免疫，为利用乳酸杆菌维护肠黏膜屏障提供理论基础。

猪肠黏膜损伤诱导的腹泻和病原的易感严重危害养猪业的健康发展，而肠干细胞通过自身的增殖分化替代损伤的肠上皮细胞是修复受损肠黏膜结构的的主要调控靶点。本课题首先证实筛选的乳酸杆菌能够提高仔猪空肠增殖相关基因的表达水平（c-Myc和Tcf1等），并且空肠绒毛高度和隐窝深度均显著增高；其次构建了肠道类器官和固有层淋巴细胞体外共培养系统，研究证实乳酸杆菌能够促进肠道类器官的生长发育并显著改善肿瘤坏死因子α引起的肠道类器官损伤；然后通过动物试验证实乳酸杆菌能够定植在肠黏膜，改善硫酸葡聚糖和沙门氏菌引起的肠黏膜损伤，改善结肠炎引起的体重下降，结肠长度变短等症状，乳酸杆菌修复肠黏膜损伤的过程中伴随着Lgr5+肠道干细胞以及潘氏细胞数量的增多；并且进一步证实乳酸杆菌能够代谢产生吲哚-3-甲醛，激活仔猪和小鼠LPLs表面的芳香烃受体诱导其表达白细胞介素22,激活STAT3信号通路促进肠道干细胞的增殖进而维护肠黏膜屏障；根据上述研究的新发现，通过原核表达成功获得了重组猪白介素22，并证明纯化的IL-22可以活化STAT3信号通路，并且在缓解肠上皮细胞凋亡和抵抗产肠毒素大肠杆菌感染。项目完成课题预期计划，发表SCI论文6篇（本基金号标注），最高影响因子大于8，影响影子大于5分的3篇，其中一篇论文发表于Cell Death and Differentiation，并被F1000Prime推荐引用，获得国际同行高度认可；申请专利2项；主持人就本项目研究成果参加高水平学术会议4次；培养3名硕士研究生。本项目将揭示乳酸杆菌调控猪肠干细胞的增殖分化并修复受损肠黏膜结构的作用机制，保障营养物质吸收和黏膜免疫，为利用乳酸杆菌维护肠黏膜屏障提供理论基础。