DLG1-PI3K信号途径通过调控CD4+IL-17+T细胞亚群分化参与克罗恩病发生及其机制研究

Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract with disturbances in immunoregulation.The pathogenesis of CD remains unclear. It is thought that helper T cell subsets participate in CD, and Th17 cell is a kind of effect cell of important functions. Recent research reports that a signaling pathway of DLG1-PI3K may regulate differentiation of Th cell subsets. We have reported DLG1 as a potential susceptibility gene for CD in the Chinese population for the first time, and have detected higher expression of DLG1 (a member of the membrane-associated guanylate kinase protein family) in CD patients compared with normal controls. We speculate that the DLG1-PI3K signaling pathway may take part in pathogenesis of CD via abduction of CD4+IL-17+T cells differentiation (including Th17 cell and IL-17+Tregs). By using the models of DLG1 knockout mice and trinitrobenzene sulphonic acid (TNBS)-induced colitis mice, and applying PI3K inhibitor, this project intends to confirm the impact of DLG1-PI3K signaling pathway on differentiation and transformation of CD4+IL-17+T cells, to clarify the effect and mechanisms of DLG1 gene in immunopathogenesis of CD. The project will provide an important experiment basis for therapy targeting DLG1 from a new perspective to explore the pathogenesis of CD.

克罗恩病（CD）是一类免疫相关肠道炎症疾病，其发病机制尚未阐明，但已发现某些Th细胞亚群参与CD发病，尤其Th17细胞（通过分泌IL-17）是介导CD发生的重要效应细胞。近期文献报道，DLG1-PI3K信号通路可调控Th细胞亚群分化。申报者前期研究也首次发现：CD患者体内DLG1（属膜相关鸟氨酸激酶级联蛋白家族成员）表达水平升高，可能是CD的潜在易感基因。申报者推测：DLG1-PI3K信号途径可能通过诱导IL-17+T细胞（包括Th17细胞、IL-17+Treg）分化而参与CD发病。为此，本项目拟建立野生小鼠CD及敲除DLG1基因的小鼠CD模型，应用PI3K抑制剂，探讨DLG1-PI3K通路对IL-17+T细胞亚群分化及转化的影响，以阐明DLG1参与CD发病的作用及其机制。本项目从新的角度探讨CD发病机制，可望为探索CD治疗的新靶点提供重要实验依据。

在小鼠体内，产生Th17细胞的T细胞和参与自身免疫的调节T细胞(Treg)在人类炎症疾病的发病机制中都发挥重要作用。在黏膜表面，Th17细胞被认为可以保护宿主免受感染，而Treg细胞则可控制宿主的免疫反应和由寄生菌群引发的炎症。克罗恩病以慢性肠壁穿透性炎症为特征，2、4、6-三硝基苯磺酸(TNBS)诱导的小鼠模型接近克罗恩病患者的肠壁炎性特征，因此该动物模型在CD研究中得到了广泛的应用。在此，我们构建TNBS诱导的炎症性肠病小鼠模型，探讨DLG1基因的下调是否会影响小鼠的T细胞功能。我们发现TNBS诱导的小鼠肠上皮层、黏膜下层和固有层受到损伤，而DLG1基因敲除的TNBS诱导的炎症性肠病小鼠肠上皮层、黏膜下层和固有层的损伤更为严重。RT-PCR、ELISA及流式细胞分析实验结果显示: DLG1基因下调使炎症因子IFN-γ、IL-17、 IL-23和Il - 6的表达明显增加，而IL- 2、IL- 10和TGF-β的表达下降，促进了T细胞分化成CD4+ CD25+ Treg细胞。免疫组化实验结果也显示了DLG1基因下调的TNBS诱导炎症性肠病小鼠模型肠黏膜中CD4、CD25、IL-17表达显著升高，Foxp3表达显著下降；Western Blot 实验显示了PTEN和 Foxp3的蛋白表达量显著降低，而RORγt蛋白水平显著表达增加。这些结果表明，DLG1基因可通过调节T细胞分化参与克罗恩病的发生发展，DLG1可能是一个潜在的抑制CD患者疾病发生发展的治疗靶点。

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