iPSC来源的MSC移植促进克罗恩病黏膜愈合及其机制

Crohn’s disease (CD) often presents in early age and commonly manifests with chronic relapsing debilitating symptoms and severe complications. With continuously increasing incidence in China, CD becomes one of the most common diseases in the gastroenterology field. However, no cure for CD until now and the current treatments are difficult to achieve and maintain remission, which results in up to 80% CD patients need for abdominal surgeries for severe complications. Therefore, it remains a great need for new therapies that are both effective and safe. Recent studies have shown that the transplantation of Mesenchymal Stem Cell (MSC) is an alternative therapeutic option to CD, but the characteristics of the MSCs including replicative senescence causing losing stem cell capacity need to be solved before application in the clinical therapy. Induced Pluripotent Stem Cell (iPSC)-derived MSCs is expected to overcome these disadvantages. Our preliminary research has shown that the iPSC-derived MSCs has stronger proliferative capacity and are more effective in colitis repair. We found that the improvement of intestinal mucosal healing is accompanied by the rebuilt of Treg/Th1/Th17 balance, the increase number of regulatory B cells (Bregs) and the change of miRNA network which leads to the improvement of epithelial barrier. Furthermore, we previously identified that MSC-activated PRRs-Notch-STAT5 axis may be involved in the activation of intestinal epithelial stem cell (IESC). Mucosal healing in CD is a tightly controlled process associated with suppression of inflammation and improvement of intestinal barrier function. It is also dependent on the balance of migration, proliferation and functional differentiation of IECs adjacent to the injured area. Based on our previous data, we proposed to test the hypothesis that iPSC-derived MSCs promotes mucosal healing in CD through the mechanism by which Bregs mediates the rebuilt of Treg/Th1/Th17 balance, miRNA network regulates epithelial barrier and inflammatory microenvironment activates IESC by PRRs-Notch-STAT5 axis. We will test our hypothesis by applying iPSC-derived MSC in multiple research models including clinical CD patients’ intestinal samples, trinitrobenzesulfonic acid (TNBS)-induced colitis mice models and the murine-derived intestinal organoids and then analyzing the regulation of iPSC-derived MSC in immunomodulation, epithelial barrier and activation of IESC. We aim to delineate the mechanism of iPSC-derived MSC in promoting mucosal healing. As such, the anticipated results will provide novel therapeutic approaches for CD, explore new target of therapeutic intervention and improve the clinical outcomes of CD patients.

我国克罗恩病(CD)发病率逐年升高，疗效不理想，致残率高。间充质干细胞(MSC)为CD治疗带来新希望，iPSC来源的MSC(iPSC-MSC)能克服MSC存在的复制性衰老、干性丢失等缺陷。我们前期研究发现iPSC-MSC可修复结肠炎小鼠黏膜损伤，同时体内Breg上调、T细胞亚群失衡纠正，miRNA网络改变、黏膜屏障修复及Notch通路激活。肠黏膜愈合是CD治疗的新目标，而黏膜免疫平衡、屏障修复及肠上皮干细胞激活是黏膜愈合的三大关键环节。基于此，我们提出假说：iPSC-MSC通过Breg细胞改善肠道炎症微环境，进而通过miRNA-紧密连接相关基因重建肠黏膜屏障，并通过PRRs-Notch-STAT5轴活化肠上皮干细胞，从而促进肠黏膜愈合。本研究以结肠炎小鼠及微小肠模型为研究载体，进一步证实iPSC-MSC促进CD黏膜愈合的效果并系统研究其机制，为临床治疗CD的转化应用提供依据。

克罗恩病(CD)是一种复发性难治性慢性肠道炎性疾病，治疗手段有限。组织来源的间充质干细胞（MSC）扩增能力有限，人诱导多能干细胞（iPSC）来源的MSC扩增能力强，具有广泛应用前景。.本课题针对CD肠黏膜愈合三大核心环节：免疫调节、黏膜屏障修复及内源性肠上皮干细胞活化，系统研究iPSC-MSCs促进CD肠黏膜愈合的作用及其机制：.一、iPSC-MSC调节T细胞免疫的机制.首先，我们进一步从细胞表面标志物、形态学证实了iPSC-MSC细胞具有类似传统的MSC特征，接着，采用iPSC-MSC细胞及其来源外泌体治疗结肠炎小鼠模型，发现均可通过调节肠道免疫失衡、促进肠上皮的修复来达到肠黏膜愈合。iPSC-MSC除能显著下调Th1/Th17外，也能下调Th9，而我们前期研究已证实Th9通过miR21-CLDN8损伤黏膜屏障促进IBD的发生。因此，iPSC-MSC通过下调Th9进而调控黏膜屏障，减轻肠道炎症。.二、 iPSC-MSC调控黏膜屏障功能的机制.通过送检外泌体芯片，经生物信息学分析，初步筛选了MALAT1、RMRP等作为目的LncRNA。人体标本验证lncRNA MALAT1及RMRP在IBD患者中表达降低。我们重点选取MALAT1作为靶基因，进一步发现MALAT1通过ceRNA机制吸附miR-146b-5p靶向NUMB、CLDN11调控CD肠黏膜屏障促进黏膜愈合。另外，我们发现miR-146b-5p可预测CD肠道黏膜炎症活动情况，是CD疾病监测新的生物标志物。.三、iPSC-MSC活化肠上皮干细胞的机制.肠上皮稳态和损伤修复依赖肠上皮干细胞的增殖和分化。CD炎症区域较非炎症区域或正常肠上皮，成熟肠上皮细胞增殖和凋亡均有增加，且出现分化的异常，提示CD炎症微环境中肠上皮干细胞存在功能受损。我们进一步探讨iPSC-MSCs 能否调控肠上皮干细胞进而促进肠黏膜修复。发现iPSC-MSCs通过分泌TSG-6进活化CD44 促进内源性肠上皮干细胞增生参与肠炎黏膜损伤修复的机制。.临床转化.在前期研究的基础上，我们开展了一项I期临床研究，纳入14例难治性CD，经外周静脉输注MSC，目前完成随访8人，其中好转5人，除外2例患者出现一过性低热，无其他不良反应发生，初步证实了干细胞治疗的有效性与安全性。.本项目的顺利开展为干细胞治疗CD提供新的理论依据，为寻求CD新治疗方案奠定基础。

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