microRNA-17-92簇调控分化的骨髓间充质干细胞对实验性结肠炎黏膜修复的影响及其机制研究

Originally isolated from bone marrow, mesenchymal stem cells (MSCs) may home to the inflamed sites and play important roles in intestinal mucosal repair. During the past two to three decades, a series of clinical reports have demonstrated that MSCs, due to their immunoregulatory and regenerative properties, could promote clinical remission and mucosal healing (MH) in patients with refractory inflammatory bowel disease (IBD), either in autologous or allogeneic settings. Up to now, stem cell transplantation has been and considered as a promising therapy for IBD. However, how to induce stem cells to proliferate and differentiate in vitro, which way is the best route to seed them in vivo, what is the key role of stem cell in the treatment of the disease? All these questions still need to be clarified..MicroRNAs have been proved to be important for regulating the differentiation and maintaining the pluripotent state of stem cells. Our previous study showed that MSCs could be grown in vitro and differentiated into epithelial like stem cells under the mediation of growth factors (activin A, FGF2) and miR-17-92 cluster. It is presumed that miR-17-92 cluster mediated-bone marrow derived-MSCs could be applied to treat dextran sulfate sodium (DSS)-induced experimental colitis. Therefore, the mimics/ inhibitors of miR-17-92 cluster, as well as the agonists/antagonist of Wnt signaling are used to interfere the growth of MSCs, and the expression of several target genes, such as Wnt3a、Axin、CDX2、GSK-3β、E2F1、β-catenin, are detected by immunohistochemistry, Western-blot and RT-qPCR. Moreover, the effects of MSCs are evaluated on intestinal mucosal repair in DSS-induced colitis in rats. The illustration of mechanism for miR-17-92 cluster and Wnt signaling pathway in the control of stem cell proliferation and differentiation will provide more evidence for the application of stem cells in the treatment of IBD.

干细胞移植治疗炎症性肠病(IBD)已显现一定效果，但如何将干细胞在体外扩增分化并有效送达受损肠段，以及相关作用机制均有待探索。基于文献和前期研究，本研究设计为：在相应生长因子诱导和miR-17-92簇定向调控下，骨髓间充质干细胞 (MSCs)→内胚层细胞→肠上皮干细胞→类肠上皮细胞逐步分化，并利用miR-17-92簇成员mimics/inhibitors和Wnt通路激动剂/阻断剂干预分化过程，检测相关基因表达，进一步探讨MSCs向类肠上皮细胞分化的条件和机制，以及miR-17-92簇成员如何协同作用调控MSCs定向分化，最后将诱导分化的MSCs通过保留灌肠移植治疗实验性结肠炎，观察其归巢、定植以及对肠道炎症改善和黏膜溃疡修复的效果。项目拟从组织、细胞、分子水平以及动物实验层面探讨miR-17-92簇定向调控分化的MSCs对结肠黏膜修复的影响及机制，为临床合理应用干细胞治疗IBD提供依据。

炎症性肠病（inflammatory bowel disease, IBD）呈慢性复发迁延病程，在我国的发病率和患病率逐年增加，常需要终身治疗，严重影响着患者的生活质量和劳动能力。目前的药物治疗还存在许多毒副作用，远期治疗效果并不理想，且均不能达到粘膜愈合的效果，亟需开发安全有效的新疗法。干细胞移植治疗炎症性肠病已显现一定效果。.本研究成功建立了“BM-MSCs→内胚层细胞→类肠上皮干细胞→类肠上皮细胞”逐步诱导BM-MSCs 分化为类肠上皮细胞的方法，BM-MSCs可在Activin A 和纤维素母细胞生长因子2 (FGF2)的作用下转变为类肠道干细胞。miR-17 通过激活Wnt/β-catenin通路促进BM-MSCs转变为类肠道干细胞，证实了体外诱导分化的类肠道干细胞可参与修复受损的肠粘膜，改善肠道的炎症状态。本课题首次提出了miR-17-92簇成员和生长因子（Activin A、FGF2）的协同诱导方案，成功建立了体外简便高效获取有功能的类肠道干细胞的方法，为肠道功能研究或干细胞移植提供原材料。