MicroRNA-21在炎症性肠病中的表达及对CD4+ T细胞分化的调控研究

miRNAs are considered probably related to the pathogenesis of inflammatory bowel disease (IBD). Controversial results about the expression of miR-21 in IBD were reported. Furthermore, the mechanism of miR-21 involved in IBD remains to be known. Our previous study found the expression of miR-21 in colitis was upregulated. To further evaluate the expression of miR-21 in IBD, a comprehensive perspective study is designed by detecting the expression of miR-21 in the mucosa, serum and feces samples in patients with IBD. The expression level of miR-21 is further compared between patients treated with and without immunosuppressants. For mechanism study, CD4+ T cells are isolated from PBMCs of patients and spleens of mice, transfected with miR-21 inhibitor or control. After activation, the proportion of CD4+ T subtypes, Th1，Th2，Th17，Treg, is detected by FACS. The expression of representative cytokines and specific transcription factors of these CD4+ T subtypes is observed in the supernatant or cell extracts via ELISA and/or Western blot. We further study whether miR-21 promotes Th17 differentiation via SMAD7/TGF-β pathway. For in vivo study, antogomiR-21 is injected into DSS mice to inhibitor miR-21. Intestinal inflammation is compared between antogomiR-21 group and control group. Above all, this study is to investigate the expression level of miR-21 in IBD and whether miR-21 involves in IBD via regulating the differentiation of CD4+ T cells, to provide a novel IBD marker and potential alternative therapy target.

近年发现miRNAs可能参与IBD发病。MiR-21被报道在IBD中的表达存在不一致的结论。且miR-21参与IBD发病机制未明。我们前期研究发现结肠炎小鼠肠黏膜miR-21的表达升高。本研究通过检测miR-21在不同活动度的IBD患者肠黏膜，血清，粪便中的表达水平，并按是否使用免疫抑制剂分组，多层面评估miR-21在IBD中的表达水平。通过分离患者外周血及结肠炎小鼠的CD4+ T细胞，转染miR-21抑制剂/对照，不同极化条件下比较CD4+ T细胞分化为Th1，Th2，Th17，Treg的比例，各Th亚群代表性细胞因子和促分化的转录因子水平。本课题进而研究miR-21是否通过调节SMAD7/TGF-β通路促进Th17分化。最后采用antigomiR-21技术，比较实验组与对照小鼠的肠道炎症水平。从而揭示miR-21是否通过调控CD4+T细胞分化参与IBD发病，为IBD的诊治提供新的思路。

本研究通过检测IBD（UC和CD）患者不同内镜下活动度分级的肠黏膜中miR-21的表达水平，证明IBD患者肠黏膜中miR-21表达水平较对照组肠息肉患者肠黏膜中显著增高，且UC患者肠黏膜中miR-21表达水平较CD患者肠黏膜中显著增高。肠黏膜中miR-21表达水平与病变部位内镜下活动度分级呈正相关。磁珠分选小鼠脾脏的Naïve CD4+ T细胞，体外转染miR-21抑制剂/对照后进行诱导分化，发现转染miR-21 inhibitor组细胞与对照组相比，Treg分化显著增加，说明miR-21能够抑制Treg分化。我们的研究表明，miR-21在IBD中可能发挥促炎作用。本研究成功构建DSS诱导的小鼠结肠炎模型，DSS结肠炎肠黏膜中miR-21表达水平较正常对照组低，可能发挥抑炎作用；miR-21通过抑制Naïve CD4+T细胞向Treg方向的分化在IBD的炎症过程中发挥重要作用。本研究提示miR-21参与了IBD的发生发展，可能在IBD中为一把双刃剑，为IBD的诊断及治疗提供了新的思路。