巨噬细胞源性miR-223通过Exosome途径介导肠黏膜屏障功能障碍在炎症性肠病中的机制研究

It is widely acknowledged that the impairment of intestinal barrier induced by the activated macrophages serves as one of the important mechanisms for the onset of IBD. Exsomes, small membrane vesicles, are commonly regarded as a new vehicle that transports genetic material between cells. Our previous studies mainly focused on the exploration of interaction of intestinal epithelial cells and we found that activated macrophages caused the dysfunction of intestinal barrier and secretion of an aberrantly elevated level of exsomes. We also demonstrated that various miRNAs were overexpressed in those exosomes through chip, among which miR-223 was observed with the largest accumulation. Based on those data, we raised a hypothesis that activated macrophages could secrete exosomes transporting miR-223 to the intestinal epithelial cells, which might regulate the function of intestinal barrier during the pathogenesis and progression of IBD. In this study, we aimed to detect whether activated macrophages could secrete exosomes transporting miR-223 to intestinal epithelial cells and explore how miR-223 impacted the intestinal barrier and microbiota. Besides, the signaling pathways and targeted proteins related to permeability would also be analyzed. Finally we will verify the relations between miR-223 and IBD in biopsy tissues from IBD patients. We believe that this study would uncover the important role of exosomal miR-223 in IBD and provide theoretical basis for the investigation of specific mechanisms and development of potential therapeutic pathway against IBD.

巨噬细胞活化导致的肠黏膜屏障功能障碍是IBD的重要机制之一。外泌体是细胞分泌的微小囊泡，近来被发现可作为信号分子在细胞间行使信息交流功能。本课题组长期研究肠黏膜上皮细胞之间的相互作用，前期结果发现：活化的巨噬细胞可介导IBD肠黏膜屏障功能障碍；巨噬细胞活化释放外泌体的数量明显增加；经芯片分析发现外泌体内多种miRNA上调，以miR-223最为显著。据此推测:巨噬细胞活化后通过分泌外泌体，传递miR-223，调控肠黏膜屏障，影响IBD的发生发展。在本研究中，拟证实巨噬细胞在活化后可分泌富含miR-233的外泌体，验证miR-223对肠黏膜屏障功能和微生态的影响，寻找miR-223通过何种下游靶蛋白影响肠黏膜屏障功能，并在IBD患者肠道组织样本中验证miR-223与IBD的关系。本课题有望发现外泌体miR-223在IBD中的重要作用，并为IBD发生发展机制的探索及治疗途径提供新的实验依据。

项目背景：外泌体是细胞间相互作用的有效媒介，其携带许多调控分子，包括miRNA，这些分子在不同的生物过程中可以发挥关键作用。到目前为止，暂未有相关研究报道巨噬细胞的外泌体参与炎症性肠病（IBD）的发展。本研究探究了IBD中巨噬细胞外泌体的特定miRNAs以及潜在的分子机制。.方法：用硫酸右旋糖酐钠（DSS）来建立IBD小鼠模型。收集脂多糖（LPS）刺激及对照的小鼠骨髓巨噬细胞（BMDMs）的上清液进行提取外泌体进行miRNA测序。通过慢病毒调控miRNA的表达，进一步研究巨噬细胞来源的外泌体miRNA的作用。在体外实验中，小鼠和人类器官通过Transwell系统与BMDMs共同培养作进一步研究巨噬细胞外泌体对肠黏膜屏障的影响。.结果：本课题发现LPS诱导的巨噬细胞可以释放含有各种miRNA的外泌体，加重IBD。我们通过分析巨噬细胞释放外泌体的miRNA测序结果结合文献，选择miR-223进一步研究。在体内，高表达的miR-223的外泌体促进了肠道屏障功能障碍的加剧，这在小鼠和人类结肠器官中得到了进一步验证。此外，对DSS诱导的结肠炎小鼠组织的mRNAs进行时间依赖性分析，结合miR-223的靶基因预测来选择候选靶向基因，并确定了屏障相关因子TMIGD1座为进一步研究对象。.结论：综合来看，本研究数据表明，巨噬细胞分泌外泌体包含miR-223，其在DSS诱导的结肠炎进展中通过抑制TMIGD1在肠道屏障功能障碍中发挥了重要作用。