Th9/IL-9通过miR-21调节Claudin-8影响肠黏膜屏障在克罗恩病发病中的作用

Impaired intestinal barrier function caused by immune-mediated inflammation is the major pathogenesis of Crohn's disease (CD).Our previous studies revealed that new T helper subset (Th9) may be invovled in the immune imbalances of CD, but the mechanism remains unclear;the inflammatory cytokines which regulate the intestinal barrier through miRNAs may be the critical pathway. We further stimulated the intestinal epithelial cell line by IL-9 and found that the expression of claudin-8 decreased, while the expression of miR-21 which targeted the 3'-UTR of claudin-9 increased. Therefore, we hypothesis that Th9/IL-9 may regulate claudin-8 through miR-21 and affect barrier funciton. To test this hypothesis, we will analyze the expression of Th9/IL-9 in CD patients and its clinical significance. In vitro and in vivo studies using experimental colitis mice will be conducted to clarify the mechnisms of Th9/IL-9 regulating the intesinal barrier through miR-21-claudin8 pathway and its role in the mucosal inflammation of CD. Multiple techniques including immunohistochemistry, luciferase analysis, Q-PCR, Western blot, transfection and epithelial permeability assay will be employed and IL-9 konck out mice will be used to further confirm this hypothesis. Therefore, our project will provide further understanding for the role of Th9/IL-9 in the pathogenesis of CD and inspire a novel potential therapeutic target for the treatment of CD patients.

免疫炎症导致肠黏膜屏障功能破坏是克罗恩病（CD）发病的核心环节。我们前期研究显示：新的辅助性T细胞亚群Th9/IL-9与CD发病相关，但机制未明；而免疫炎症通过miRNA调控黏膜屏障功能可能是其关键途径。我们进一步采用IL-9刺激肠上皮细胞，发现Claudin-8(CLDN8)表达下调，作用于CLDN8 3'-UTR区的miR-21则上调。因此，我们推测Th9/IL-9可诱导miR-21表达变化靶向作用于CLDN8从而影响黏膜屏障。基于此，我们拟分析CD患者Th9/IL-9的表达及其临床意义；通过体外细胞学实验及体内动物实验，采用免疫组化、荧光素酶试验、定量PCR、蛋白印迹、细胞转染及上皮通透性测定等技术阐明Th9/IL-9可调控miR-21靶向作用于CLDN8从而导致CD肠道黏膜屏障损伤的机制；并通过IL-9基因敲除动物模型进一步验证，为明确Th9/IL-9在CD发病中的作用提供依据。

免疫炎症导致肠黏膜屏障功能破坏是克罗恩病（CD）发病的核心环节，探讨免疫与肠上皮粘膜屏障之间的相互作用有助于发现新的治疗靶点。我们在前期研究基础上提出研究假说：新的辅助性T细胞亚群Th9/IL-9可诱导miR-21表达变化靶向作用于CLDN8从而影响黏膜屏障。为了验证研究假说，我们分析CD患者IL-9的表达；通过体外细胞学实验、体内动物模型（包括IL-9处理及抗IL-9处理的小鼠结肠炎模型）进行研究。结果发现：1.CD患者结肠IL-9的表达高于正常对照，而CLDN8与其表达呈负相关；2.IL-9可加重小鼠结肠炎，而抗IL-9可减轻小鼠结肠炎，同时伴随CLDN8的变化；3.miR-21介导了IL-9-CLDN8之间的相互作用。研究结果与我们预期相符，已发表相关SCI论著1篇，另有2篇论著投稿中，培养博士研究生1名，基本完成了预期的指标。该研究阐明了IL-9-miR21-CLDN8通路在CD中的作用，可作为临床生物标志物预测药物疗效及评估疾病情况，且可能用于新药研发。