VEGFR-3介导的淋巴管生成在实验性炎症性肠病发病中的作用和对慢性炎症的调控研究

Inflammation bowal disease(IBD) is a group of intestinal chronic inflammatory disease, with increasing incidence and tough therapy,but its pathogenesis has not been clarified. Recently, lymphatic system is reported to involved in inflammatory response,and may play a crucial role in the pathogenesis of IBD. However, its mechanism is unclear now. Vascular endothelial growth factor receptor(VEGFR)-3 signaling is the main molecular mechanism regulating lymphangiogenesis,but its effect on IBD has not been analyzed now.The previous studies mainly focus on the mechanism of how the chronic inflammation "IN" the intestines,however,in this study from a new point of view, we aim to investigate the mechanisms of how the chronic inflammation "OUT" of the intestines regulated by inflammatory lymphangiogenesis. The ulcerative colitis (UC) and Crohn's disease (CD) experimental models in rats using trinitrobenzene-sulfonic acid/alcohol are established.Then,overexpression of VEGF-C and -D lentivirus vectors are constructed to induce lymphangiogenesis via VEGFR-3 signaling in the experimental IBD rats by intra peritoneum. Meanwhile,neutralizing anti-VEGFR-3 monoantibody(mAb) is used to block the signaling, and these two effects compared. The effects of anti-TNF-α mAb,dexamethasone,and VEGF-A/VEGFR-2 signaling in experimental IBD models are as the controls. During the devolpment of the experimental IBDs, we analyze the numbers of lymphatic vesels (lymphangiogenesis),the funtion of leucocytes trafficing through lymphantics to regional lymph nodes, tissue edema controling,bacterial and chemokine clearance.The role of inflammatory lymphangiogenesis via VEGFR-3 on controling chronic inflammation in the pathogenesis of experimental IBD are investigated. And the molecular mechanisms of VEGFR-3 governing lymphangiogenesis may provide new possibilities to treat IBD.

IBD是一组肠道慢性炎症性疾病，发病率日益上升，临床治疗困难，但病因与发病机制却未完全明确。近年发现淋巴管系统参与了炎症反应，VEGFR-3通路是调节淋巴管生成的主要机制，但淋巴管生成在IBD发病过程的作用却不清楚。既往研究大多集中在IBD时炎症如何"进入"肠道，本项目从调控肠道淋巴管生成和功能的新角度出发，研究如何使慢性炎症"离开"肠道的机制。拟分别建立TNBS/乙醇UC和CD大鼠模型，对比分析过表达VEGF-C、-D慢病毒载体激活VEGFR-3通路，和中和性抗体阻断该通路后的不同效应，以抗TNF-α抗体、地塞米松和VEGF-A/VEGFR-2通路的作用为对照，观察肠道淋巴管生成、淋巴管引流功能、炎症变化、炎性细胞因子和肠道细菌清除的变化，探讨VEGFR-3调节淋巴管生成对IBD发病过程的影响及对慢性炎症的调控，阐明淋巴管在IBD发病机制中的作用及VEGFR-3靶向治疗IBD的潜在价值。

1.背景：炎症性肠病(IBDs)是一组病因和发病机制未完全明确、复发缓解交替和治疗棘手的肠道慢性炎症性疾病。近来一种新的观点指出IBD可能是一种“管道性疾病”，其中肠道淋巴管系统具有调节炎症“离开(OUT)”肠道的机制备受关注。本课题即针对肠道淋巴管生成在急慢性肠炎中的作用及其调节肠道炎症的机制进行研究。 .2.研究内容：分别建立5%DSS诱导的小鼠急性结肠炎和2%DSS诱导的慢性结肠炎模型。通过调节淋巴管生成最重要的VEGF-C/VEGFR-3途径，用高表达VEGF-C的腺病毒/重组蛋白行刺激实验，抗VEGFR-3抗体进行阻断实验，观察调节VEGFR-3通路对急慢性肠炎中淋巴管密度(LVD)、功能、结肠炎症、肠壁水肿、淋巴管生成因子和促炎因子表达的变化关系及与EKR通路的关系。.3.重要结果与关键数据：(1) 刺激实验中，肠道高表达VEGF-C使急性结肠炎小鼠的肠道炎症、肠壁水肿、LVD (P<0.001)、淋巴管直径(P< 0.001)以及VEGFR-3mRNA表达均明显增加(相关论文已被录用)；而慢性结肠炎小鼠的结肠炎症和组织学损害均减轻 (P< 0.05)、淋巴管明显扩张(P=0.016)、淋巴管引流增强及结肠VEGF-C蛋白浓度增加，但LVD无增加、结肠组织ERK1/2、p-ERK1/2蛋白表达无上调（P>0.05）。急性与慢性结肠炎对增强VEGFR-3通路的反应不同，且ERK/p-ERK通路未参与VEGF-C/VEGFR-3通路调节的小鼠慢性结肠炎症。(2) 在注射抗VEGFR-3抗体的阻断实验中，急、慢性结肠炎小鼠的肠道炎症均加重，淋巴引流功能下降(急性: P=0.0344; 慢性: P=0.025)、肠壁水肿、炎性细胞浸润和细胞因子表达增加(P<0.05)。但慢性结肠炎中淋巴管明显扩张扭曲(P=0.0328)且LVD减少(P=0.012)；而急性结肠炎中淋巴管虽明显扭曲扩张 (P=0.0437)，但LVD无改变。表明阻断VEGFR-3通路均可造成急、慢性结肠炎中淋巴管引流功能下降从而使肠壁炎症加重，但与慢性炎症不同，急性炎症中未影响淋巴管生成(相关论文已被录用)。.4.科学意义：在急、慢性结肠炎中刺激或阻断VEGFR-3途径导致淋巴管数量及其功能出现不同的变化，进而影响肠道炎症。这表明在肠道炎症时，靶向VEGF-C/VEGFR-3途径促进淋巴管