miR-124介导Th17/Treg细胞分化失衡在克罗恩病发病中的作用机制

Imbalance of Th17/Treg cell differentiation results in autoimmune diseases including Crohn's disease(CD). However, the detailed mechanism remains unknown. Our previous study also verified that there was an imbalance of Th17/Treg in patients with active CD.We firstly found that miR-124 was overexpressed in inflamed human tissues. Aryl hydrocarbon receptor(AhR) was the target gene of miR-124, which regulated Th17/Treg cell differentiation and relevant immune responses.Based on these, our hypothesis is that miR-124 mediates Th17/Treg cell differentiation imbalance by inhibition of the AhR expression, eventually leading to intestinal inflammatory formation. This project intends to use the technology of lymphocyte directed differentiation in vitro,miRNA lentiviral vector construction，reporter gene assay, and so on to investigate the role of miR-124 in the formation of CD and keeping the balance of Th17/Treg differentiation; overexpression and silencing of miR-124 to observe alterations in AhR expression and Th17/Treg differentiation; furthermore, overexpression and silencing of AhR gene to verify miR-124 mediated Th17/Treg cell differentiation imbalance by inhibition of the AhR expression. This helps to clarify the molecular mechanism of Th17/Treg differentiation imbalance in CD pathogenesis, and provide a potiential target for the treatment of CD.

Th17/Treg细胞失衡介导自身免疫性疾病包括克罗恩病（CD）的发生，导致Th17/Treg失衡机制尚不清楚。我们前期研究发现：CD患者存在Th17/Treg失衡；miR-124在病变部位分离的淋巴细胞中呈高表达；其靶基因为芳香烃受体(AhR)，AhR在病变部位低表达；而AhR是调控Th17/Treg分化重要因子。因而我们提出miR-124靶向抑制AhR表达，介导Th17/Treg细胞失衡，肠道炎症形成的科学假说。本课题拟利用淋巴细胞定向分化，miRNA载体构建，荧光素酶报告实验等手段，获得miR-124参与CD发病及其与Th17/Treg失衡相关依据；过表达及沉默miR-124，获得Th17/Treg分化平衡与AhR改变的依据；进一步过表达及沉默AhR，论证miR-124介导Th17/Treg分化平衡的AhR调控机制，阐明CD发病中导致Th17/Treg失衡分子机制,为CD防治提供靶点

项目背景：Th17/Treg细胞失衡在克罗恩病（CD）的发生发展中发挥重要作用，导致Th17/Treg失衡机制尚不清楚。我们前期研究发现：CD患者病变肠组织miR-124高表达，芳香烃受体(AhR)低表达；AhR是miR-124靶基因，而AhR是调控Th17/Treg分化重要因子。因而我们提出miR-124靶向抑制AhR表达，介导Th17/Treg细胞失衡，肠道炎症形成的科学假说。.主要研究内容：通过临床相关研究，获得miR-124参与CD发病及其与Th17/Treg细胞失衡相关临床依据获得；过表达或表达沉默miR-124观察对AhR表达以及Th17/Treg细胞分化平衡影响；荧光素酶实验进一步确定AhR是miR-124的靶基因；进一步过表达及沉默AhR，论证miR-124介导Th17/Treg分化平衡的AhR调控机制，阐明CD发病中导致Th17/Treg失衡分子机制。.重要结果、关键数据及其科学意义：.1.荧光定量PCR检测miRNA-124在CD患者肠组织中表达，发现miR-124在活动期CD患者病变肠组织高表达，miRNA原位杂交检测发现miR-124在病变肠组织CD4阳性细胞及肠上皮细胞中高表达，提示miR-124参与了CD患者疾病过程。.2.流式细胞检测结果显示活动期CD患者外周血Th17细胞增加，Treg细胞减少。3.ELISA法检测活动期CD患者血清Treg及Th17相关细胞因子，结果显示：与对照组相比，活动期CD患者血IL-17水平明显升高, 而IL-10及TGF-β减低。.4. 免疫荧光及Western blot检测肠黏膜组织AhR及Treg细胞特异分化因子FoxP3表达，结果显示：CD患者病变肠黏膜组织AhR减少，而FoxP3表达降低。.5.选取Caco2细胞，过表达或表达沉默miR-124，发现miR-124高表达，AhR表达减低，反之，AhR表达升高。.6. 构建了miR-124过表达小鼠，予TNBS造模，以聚醚酰亚胺（PEI）为载体，将其与anti-miR-124混合后给小鼠灌肠，发现高表达miR-124的转基因小鼠TNBS造模后肠道炎症明显重于WT小鼠，给予anti-miR-124后肠道炎症减轻。.以上结果提示miR-124靶向抑制AhR的表达，影响Th17/Treg细胞分化平衡，参与肠道炎性损伤过程。