益生菌通过促炎症因子调控Wnt/β-catenin信号通路维持溃疡性结肠炎缓解的机制研究

The Maintenance of remission of Ulcerative Colitis (UC) and prevention of carcinogenesis are the focus of clinical concern. Clinical research found that probiotic has satisfactory curative effect on those respects, whereas the mechanism is not quite known. We had found the existence of disturbance of intestinal flora and abnormal expression of inflammatory factors before, which is probably related with the influence of pathogenic bacteria toxin to relevant signal pathway after the disturbance of intestinal flora, through the method of genomic sequencing and microarray. On this basis, we put forward the hypothesis that probiotic may control inflammation and prevent carcinogenesis through regulating the inflammatory factors and down regulation of the Wnt signal pathway. Our project aims at : 1.On DSS mice UC model, discuss the influence to the genesis of inflammation and carcinoma after intervention of probiotic，and detect the expression of IL-6、IL-10、TNF-α、Stat3、NF-kb mRNA and protein, as well as distribution of β-catenin in cells. 2. Co-culture of normal colon cell strain CDD841 and tumor cell strain HT116 with probiotic to detect relationship between disease and the above-mentioned proteins. 3. Treatment of UC patients using probiotic, observe the clinical course and the relationship with the previous items. Our project will provide evidence for the maintenance of remission of UC and prevention of carcinogenesis.

溃疡性结肠炎（UC）维持缓解和预防肿瘤发生是临床关注的焦点，目前临床研究发现益生菌在这些方面具有较好的疗效，但确切机制尚未明确。我们前期在DSS小鼠UC模型中发现存在肠道菌群紊乱、促炎症因子表达异常，通过元基因组测序及表达谱芯片发现可能与肠道菌群紊乱后致病菌毒素参与影响相关信号通路有关。在此基础上，我们提出益生菌可能通过调节促炎症因子，下调Wnt途径，控制炎症和防止肿瘤形成这一假设。本课题拟：1在DSS小鼠动物模型中，探讨益生菌干预后对炎症和肿瘤发生的影响，检测IL6、IL10、TNF-α、Stat3和NF-kB mRNA和蛋白表达，以及β-catenin在细胞内的分布；2 结肠正常细胞株CDD841和肿瘤细胞株HT116与益生菌共培养，检测上述蛋白的表达及相关信号途径；3 在UC患者中给予益生菌后观察菌群改变及上述蛋白与疾病的关系。本课题将为临床UC维持缓解和预防癌变的治疗提供理论依据。

项目背景：溃疡性结肠炎（UC）癌变是UC的严重并发症。随着UC发病率的日益增高，UC癌变越来越多的引起临床的重视。既往研究表明，肠道菌群失衡在溃结炎症和肿瘤发生过程中具有重要作用，益生菌可抑制癌变小鼠肿瘤形成，但并未对机制及肠道菌群变化进行探讨。.主要研究内容：1.探索益生菌对AOM/DSS诱导的UC癌变小鼠模型的干预作用及相关机制。2.分析益生菌VSL#3、布拉氏酵母菌干预对AOM/DSS诱导的溃疡性结肠炎癌变模型小鼠成瘤的影响及小鼠粪便和黏膜菌群的变化，探讨肠道菌群变化在UC癌变中的作用。3.益生菌与细胞共培养，检测对Wnt通路的影响。4.分析不同病变范围UC患者肠道菌群差异。.重要结果及关键数据：1.益生菌对溃疡性结肠炎癌变的抑制作用及相关机制初探：C57BL/6小鼠随机分为5组，分别为空白对照组、建模对照组、5-ASA灌胃组、布拉氏酵母菌灌胃组、VSL#3灌胃组。实验结束处死小鼠后发现，与建模对照组比较，各灌胃组瘤负荷、小鼠结肠组织炎症因子水平、NF-κB转录活性、TCF-4转录活性均显著下降，β-catenin细胞核表达量减少。2. 益生菌对溃疡性结肠炎癌变的抑制作用及菌群分析：粪便菌群分析显示模型对照组较空白对照组乳杆菌属减少，颤杆菌属、Lachnoclostridium增加；益生菌干预后，芽孢杆菌属、乳杆菌属增加，颤杆菌属、拟杆菌属减少。黏膜菌群分析显示：模型对照组较空白对照组双歧杆菌属减少，拟普雷沃菌属增加。UC癌变小鼠肠道菌群紊乱，黏膜和粪便菌群均表现为有益菌属减少、有害菌属增加，补充益生菌有益于UC癌变小鼠粪便和黏膜菌群实现再平衡。3.益生菌沉淀与细胞共培养，检测对Wnt通路影响，影响环节与实验动物结果一致。4. UC患者粪便菌群生物多样性降低，随着病变范围增大，患者粪便和肠黏膜菌群均表现为有益菌属减少，有害菌属增加。患者黏膜菌群的改变呈全结肠分布改变特点。.科学意义：本研究有助于了解UC癌变过程中炎症和肿瘤发生之间的相关机制，对肠道菌群在UC癌变中的作用有更加深入的认识，并为益生菌制剂预防UC癌变提供有力的理论支持。