TLRs配体活化间充质干细胞在克罗恩病非纤维化粘膜愈合中的作用及其机制

Intestinal fibrosis represents the biggest challenge for the functional healing of intestinal mucosal barrier injured in Crohn's disease. Till now, the available treatment tools could not prevent or reverse intestinal fibrosis. It has been proven both in animal model and clinical trial, mesenchymal stem cells (MSCs) with immune regulatory and regenerative functions, may serve as an effective treatment strategy for CD, with the promising of promoting function repair. However, the function of MSCs mainly depends on the surrounding microenvironment. Our preliminary study has found that intestinal mesenchymal stem cells isolating from CD patients demonstrate abnormal self-proliferation and immunomodulatory capacity when compared with normal controls. The intestinal microenvironment of CD patients may not be suitable to prime and polarize MSCs to the required functional status. Thus how to prime MSCs in vitro to exert the function of mucosal healing and anti-fibrosis in vivo may represent the key technique problem when applied in the treatment of CD. We further confirmed that in vivo TNBS-induced intestinal colitis animal models, TLRs ligands priming entailed MSCs more powerful immunomodulatory functions, ameliorated both symptomatic and historical scores, and induced the differentiation of T cells from colitis mesenteric lymph nodes into Treg. However, the attributing mechanism is still unknown, which may partly be due to the secretion of anti-inflammatory and anti-fibrosis cytokines. And whether such polarized MSCs following TLR ligands priming may vary the naive MSCs for functional healing of injured mucosal of CD has not been reported. In the present study, we plan to explore the role of differential priming MSCs with TLRs ligands for the functional healing of intestinal mucosal barrier injured in CD. The main contents include: (1) to explore whether polarized MSCs is more controllable than the naive MSCs for promoting functional (non-fibrotic) healing of chronic injured mucosal barrier by cytology in vitro and in vivo animal studies; (2) to explore the potential role of signaling pathway of TLR3/4-Jagged-1-Notch. In this study, in new point of view of differential priming MSCs through TLRs ligands, we aim to lay the foundation for functional healing of injured mucosa of CD.

肠壁纤维化是克罗恩病（Crohn's disease, CD）治疗中实现粘膜功能性修复的最大障碍,现有治疗手段均无法阻止肠壁纤维化。动物及临床研究提示间充质干细胞（MSCs）移植可有效治疗CD，有望实现功能性修复，但其作用受微环境影响。我们前期研究发现CD患者肠MSCs存在自我增殖及免疫调节异常，其局部微环境不适合MSCs获取治疗所需的功能状态。如何诱导活化MSCs发挥粘膜修复及抗纤维化作用是MSCs移植治疗CD的关键技术问题。我们在动物模型体内证实，TLRs配体极化后的MSCs抗炎及抗纤维化细胞因子上调，显著改善症状及组织学评分，并诱导CD4+T细胞向Treg细胞分化，但机制不明。本项目拟从体外细胞学及动物体内研究阐明TLRs配体活化后MSCs如何发挥更可控的促粘膜愈合及促非纤维化修复作用；同时探索TLRs-Jagged1-Notch信号通路的可能作用，为实现CD粘膜功能性修复奠定基础。

背景：肠道长期慢性炎症反复发作过程中产生的肠壁纤维化是克罗恩病（Crohn’s disease, CD）治疗中实现粘膜功能性修复的最大障碍,现有治疗手段均无法阻止肠壁纤维化。目前针对狭窄治疗的手段如内镜球囊扩张术、狭窄成形术和手术切除等仅是对症治疗，复发率高，因此，研究如何阻断肠壁的纤维化进程对于临床治疗尤为重要。.目的：本研究旨在明确Toll样受体（TLRs）配体预处理极化后的人脐带间充质干细胞（hUC-MSCs)对慢性炎症诱导的小鼠肠壁纤维化的治疗作用。明确其可能机制。 .方法：采用6-8周龄BALB/c雄性小鼠，通过连续6~8周递增剂量重复TNBS的灌肠制作CD肠道狭窄小鼠模型。在造模后2小时经腹腔注射不同TLRs配体活化的hUC-MSC，观察生存率、结肠炎表现及组织学评分评价疗效。为了明确其机制，通过体外细胞增殖实验，体内结肠炎模型，研究siRNA干扰TLR3受体，DAPT阻断Notch-1信号通路后hUC-MSCs的作用。.结果：通过连续6~8周递增剂量重复TNBS的灌肠可致小鼠结肠肠壁产生纤维化，以透壁性炎症、肠道狭窄为特征。CD小鼠肠道狭窄伴随肌成纤维细胞的激活，表现为肌成纤维细胞(vimentin+, α-SMA+)及成纤维细胞(vimentin+, α-SMA-)在固有层及粘膜下层表达增加。通过免疫荧光染色，提示TNBS诱导慢性纤维化小鼠肠道中TGF-β+Smad3+双阳性细胞增加； qPCR及蛋白印记法进一步证实TGF-β-Smad3通路在肠壁纤维化过程中活化。人体组织确证狭窄型CD手术标本Masson染色狭窄型CD患者肠组织大量胶原沉积，同时免疫荧光染色证实肌成纤维细胞活化（α-SMA+Ki67+）伴随TGF-β-Smad3通路的激活。进一步通过体内外实验证实经TLR配体预处理的MSC能诱导肌成纤维细胞的凋亡及抑制其增殖，降低结肠炎小鼠的死亡率、减轻症状及改善组织炎症，并通过抑制TGF-β-Smad3通路改善肠道纤维化程度。.结论：(1) 肌成纤维细胞在TNBS诱导的慢性结肠炎小鼠及狭窄型CD患者增加且伴随TGF-β-Smad3信号通路的活化。 (2)TLR3活化的MSC能诱导肌成纤维细胞的凋亡及抑制其增殖，降低结肠炎小鼠的死亡率、减轻症状及改善组织炎症，并通过抑制TGF-β-Smad3通路改善肠道纤维化程度。