派伊尔结内生乳杆菌调节粘液生物膜结构及免疫调节功能的研究

Peyer's patches (PP) is major site where commensal bacteria encounter host immune cell and to activate a cross-talk for sIgA production. Some lactobacilli inhabit within PP. Their functions are not known until now. A bulk of sIgA is located within mucus layer after secretion and selectively bind bacteria who release molecules, such as peptidoglycan and CpG DNA, to modulate immune resposne of epithelial cell in sute. We suggest a hypothesis in this study: few specific commensal lactobacilli enter animal PP, continuously induce production of natural sIgA to control distrubution of bacteria within mucus biofilm and releasing of immune modulatary molecules, and maintain defensive innate immunity of gut. We want to analyze bacteria inhabiting PP, isolate and identify major lactobacilli strain, and study the mechanism they enter PP. Specific strain would be used to treat mice for studying their internalization into PP in vivo and the effect on spatial organization of bacterial flora and sIgA in mucus biofilm. Source, distribution and diffusion of extracellular DNA and peptidoglycan will be also studied. These will be also studied in vitro in anaerobic biofilms of colonic microbiota constructed in mucin hydrogel containing different concentration of sIgA. This study would promote screening of immunoregulatory probiotic and explanation the mechansim, which is of theoretical significance and have application value in development of microecological agent.

派伊尔结（PP）是肠道共生菌与免疫活性细胞发生接触，实现"对话"诱导sIgA产生的主要部位，某些共生乳杆菌可在PP内存活，其作用尚不清楚。sIgA大部分存在于粘液层，选择性结合细菌。粘液细菌释放的肽聚糖和CpG DNA等分子调节着局部上皮细胞免疫反应。我们提出假设：少量特定乳杆菌进入动物PP，作为PP内生菌持续诱导非特异性sIgA产生，控制粘液生物膜菌群构成和免疫调节分子释放，从而调节上皮细胞先天性免疫防御反应。本课题拟分析、分离和鉴定动物PP内生乳杆菌，并探讨其内化PP的机理；用所得菌株处理小鼠，观察其进入PP的效率、对粘液生物膜sIgA和菌群构成及分布的影响，并分析生物膜肽聚糖和细菌胞外DNA含量、来源及其在粘液中的扩散行为，同时构建体外粘液生物膜以证实我们的假设，进而提出筛寻免疫调节型益生菌的新方式并阐明其作用机制，其成果在高效微生态制剂研究领域必将具有一定的理论意义和应用价

某些肠道共生乳杆菌可在派伊尔结（PP）内存活，由于PP是肠道分泌型IgA（sIgA）的主要诱导部位，这些乳杆菌可能参与调控sIgA产生。sIgA是参与形成肠道粘膜生物膜的重要分子。细菌细胞外eDNA也可能参与形成肠道生物膜，不同细菌来源的eDNA具有多样性免疫调节作用。我们提出假设：PP乳杆菌持续诱导sIgA产生，控制粘液生物膜菌群构成和细菌eDNA释放，从而调节上皮细胞先天性免疫防御反应。.本项目开展了四方面研究：1）高脂饲料(HFD)诱导肥胖对PP菌群构成的影响，及与肠道免疫反应的关系。2）小鼠PP乳杆菌的分离、鉴定和分型分析，以及HFD敏感菌株的抗肥胖效果。3）大鼠PP乳杆菌的分离，及典型菌株对正常和维生素A 缺乏大鼠sIgA产生和肠道菌群多样性的影响。4）小鼠PP和回肠特征性乳杆菌的分离、鉴定，及其对小鼠粘液eDNA和隐窝抗菌肽产生相关基因表达的影响。.本研究主要取得以下重要结果：1）大鼠和小鼠派伊尔结优势乳杆菌为罗伊氏乳杆菌（L. reuteri）。2）HFD诱导肥胖小鼠PP乳杆菌和L. reuteri显著降低。派伊尔结乳杆菌含量与回肠粘膜炎性细胞因子呈显著负相关，而与抗炎性细胞因子呈显著正相关。3）DIO小鼠PP中氧化应激耐受性弱/强L.reuteri比值显著下降，高脂敏感的菌株L. reuteri L3在体外能够促进PP细胞产生IL-10，而抑制IL-6、IL-12和TNF-α的产生，可保护PP细胞免受炎性损伤。该菌株在体内能显著抑制高脂膳食引起的体重、血糖、血胰岛素水平的增加，主要通过诱导抗炎性反应实现。4）大鼠PP L.reuteri可调控PP乳杆菌丰富度，通过维生素A依赖方式诱导sIgA产生。5）小鼠结肠粘液层存在eDNA，且主要为拟杆菌门细菌所释放。派伊尔结特异的L.reuteri 可以调控粘液eDNA构成，从而调控隐窝Myd88信号通路，及其介导的抗菌肽表达。实现对粘液菌群的回路调节。.本研究证明PP特有的L.reuteri是调节肠道免疫反应重要参与者，也证明sIgA和粘液eDNA的构成受PP L.reuteri调节，通过控制隐窝细胞抗菌肽表达维持粘液菌群稳态。该发现是动物粘膜生理学的重要补充，也为筛选新型免疫调节性益生菌提供了重要理论依据。