脂肪干细胞对实验性结肠炎肠道淋巴管生成的影响及其免疫调节机制研究

The inflammatory bowel disease (IBD) is a group diseases with unknown pathogiogenesis and difficult treatment. Our previus study showed inflammation-associated lymphangiogenesis was involved in development of acute and chronic inflammation. Vascular endothelial growth factor-c (VEGF-C) can promote lymphangiogenesis and attenuates chronic intestinal inflammation, however, acute colitis can't be reduced. . Adipose-derived stem cells(ADSCs) secrete mutiple cytokines and growth factors, having the effect on anti-inflammation and inmmunoregulation at the same time. Based on these effects, it becomes a prominsing therapy in IBD. However, its mechanism is still unknow and clincal effects are controversial. Therefore in this study, we hypothasized that VEGF-C stimulated ADSCs will have stronger effects on promoting lymphangiogenesis and increasing lymph flow drainage, combined with secretion and immunoregulation of ADSCs. That means combination with inhibition 'IN'and promotion of "OUT" of intestinal inflammation. According to this basis, we will investigate the prolifration, tube formation and migration of VEGF-C stimulated intestinal lymphatic endothelial cells (LECs) islated from C57BL/6 mice, and further detecting ADSCs secretion in vitro. In vivo study, we will establish TNBS- and DSS-induced acute and chronic colitis models to study the effects of VEGF-C stimulated ADSCs in different ways on intestinal inflammation, lymphangiogenesis, inmmunoregulations and their interaction. We are going to provide the theoretical and experimental evedences for the new point and new mechanism of lymphatic system and ADSCs in treating IBD.

炎症性肠病(IBD)是一组病因未明且临床治疗棘手的慢性肠道炎症性疾病。我们前期研究发现，炎性淋巴管生成参与了炎症发生发展。血管内皮生长因子-C(VEGF-C)可促进淋巴管引流而减轻慢性肠道炎症，但不缓解急性炎症。脂肪干细胞(ADSCs)可分泌多种细胞因子和生长因子促进内皮功能，并可抗炎和调节免疫，在细胞治疗IBD上极具前景，但疗效不一机制不明。由此我们设想，VEGF-C刺激ADSCs可更强地促淋巴管生成加强炎症引流，结合ADSCs分泌及免疫调节作用，抑制炎症“进”并炎症“出”，故可更有效地减轻肠道炎症。为此，我们将研究VEGF-C刺激的ADSCs在体外对肠道淋巴管内皮细胞增殖、管腔形成和迁移能力的影响及ADSCs的分泌功能；在体内小鼠急、慢性结肠炎模型中，研究炎症反应、淋巴管生成及免疫调节的机制及其相互作用，为阐明ADSCs和淋巴管系统治疗IBD的新角度和新机制提供理论和实验依据。

研究背景：.炎性淋巴管生成参与了炎症发生发展。脂肪干细胞(ADSCs)可旁分泌多种细胞因子和生长因子发挥抗炎和调节免疫功能，在细胞治疗IBD上极具前景，但在IBD的疗效与机制不明。已发现VEGF-C刺激的ADSCs凝胶可更有效地促进伤口愈合。由此设想，VEGF-C刺激的ADSCs可通过加强淋巴管引流功能促进炎症消退，从抑制炎症“进”并加强炎症“出”两方面更有效地减轻肠道炎症。.主要研究内容： .1.体外观察VEGF-C 刺激的ADSCs对肠道淋巴管内皮(LECs)的增殖、迁移和管腔形成的影响；.2.观察在TNBS诱导的急、慢性小鼠结肠炎模型中，VEGF-C刺激的ADSC对淋巴管引流功能的影响及对肠道炎症、淋巴细胞归巢、MLN中菌群和免疫细胞等的影响。.重要结果及数据：.1.在体外VEGF-C刺激的ADSCs可通过分泌外泌体miR-132靶向smad7调节TGF-β/Smad信号途径，促进VEGF-C依赖的LEC的增殖、迁移和淋巴管生成能力。.2.急、慢性小鼠TNBS结肠炎中，与ADSCs组相比，VEGF-C+ADSCs处理后结肠长度显著变长（p>0.05）、体重明显下降（p>0.05）；结肠细胞增殖、IL-17和TNF-ɑ mRNA水平明显下降（p>0.05）。 .3.原代分离小鼠ADSCs并鉴定和检测其诱导成骨和成脂功能。慢性小鼠TNBS结肠炎中，VEGF-C刺激后ADSC组的生存率(100%)高于ADSCs组(80%)。与ADSC组相比，VEGF-C+ADSCs组的结肠炎症、组织学评分（0.80±0.45 vs 1.80±0.45，P<0.05）、结肠细胞增殖(51.2±3.4% vs 35.3 ±8.66%，P= 0.0002)、结肠伊文思蓝含量均明显下降 P<0.0001），表明淋巴管引流功能明显增强；细胞凋亡显著受抑（21.2±3.5% vs 15.8±2.2%, P=0.0442）、LVD明显增加，促炎细胞因子(IL-6、TNF-ɑ、IFN-γmRNA)分泌明显下降，抑炎细胞因子IL-10mRNA则明显升高 (P<0.01)。.科学意义：.VEGF-C刺激优化后的ADSC比单纯ADSC具有更好的控制慢性肠道炎症的疗效，其机制与ADSC分泌外泌体miRNA增强LEC功能，并进一步加强淋巴管消退炎症的作用、减轻肠黏膜炎症有关。