miRNA-19b在克罗恩病形成中调控Th17细胞分化的分子机制

Abnormal Th17 cell differentiation mediated immune diseases, including Crohn's disease (CD) formation, the mechanism of abnormal Th17 differentiation is not yet clear.Our previous study found that: miR-19b was highly expressed in lymphocytes from peripheral blood and lesion tissue from patients with active CD； The expression of miR-19b was increased during the differentiation from Th0 cells to Th17 subset in vitro; bioinformatics prediction and fluoresceinenzyme Reporting System showed that cytokine signaling inhibitor 3 (SOCS3) was target gene of miR-19b;SOCS3 is an important negative regulatory molecule in Th17 differentiation process. This project intends to use the technology of lymphocyte directed differentiation in vitro,construction of miRNA lentiviral vector and etc. to investigate the association of miR-19b with the formation of CD and the regulation of Th17 differentiation on the basis of clinical samples research; overexpression and silencing of miR-19b to observe Th17 differentiation and SOCS3 expression; furthermore, overexpression and silencing of SOCS3 gene to investigate the mechanism of miR-19b regulating Th17 differentiation by SOCS3, and verify the regulation role of miR-19b in SOCS3 expression by luciferase reporter system. This helps to clarify the molecular mechanism of Th17 differentiation in CD pathogenesis, and provide a potiential useful target for the treatment of CD.

Th17细胞异常分化介导自身免疫性疾病，包括克罗恩病（CD）的形成，导致Th17异常分化机制尚不清楚。我们前期研究发现：miR-19b在活动期CD血及病变组织淋巴细胞中高表达；体外Th0细胞向Th17亚群分化过程中miR-19b高表达；生物信息预测及荧光素酶报告系统显示细胞因子信号抑制因子3（SOCS3)为miR-19b靶基因；而SOCS3是Th17分化中重要的负调节分子。本课题拟利用淋巴细胞定向分化，miRNA载体构建等手段，通过对临床标本研究，获得miR-19b参与CD发病及其与Th17分化相关的临床依据；过表达及沉默miR-19b，观察Th17分化及SOCS3表达；进一步将SOCS3基因过表达及沉默，论证miR-19b影响Th17分化的SOCS3机制，以荧光素酶报告系统验证miR-19b对SOCS3的调控作用，阐明在CD发病中调控Th17分化的分子机制，为CD治疗提供有益靶点。

项目背景：Th17细胞异常增多在克罗恩病（CD）发病中发挥重要作用，导致Th17异常分化的机制尚不清楚。我们前期研究发现：miR-19b在活动期CD血及病变肠组织淋巴细胞中高表达；生物信息预测细胞因子信号抑制因子3（SOCS3)为miR-19b靶基因而SOCS3是Th17分化中重要负调节因子。基于此，拟开展：miR-19b在克罗恩病形成中调控Th17细胞分化的分子机制研究。.主要研究内容：通过临床相关研究，获得miR-19b及Th17参与CD发病的临床依据；过表达及沉默miR-19b，观察对SOCS3表达及Th17分化的影响；进一步过表达及沉默SOCS3，论证miR-19b影响Th17分化的SOCS3机制，以荧光素酶报告实验验证miR-19b对SOCS3的靶向调控。.重要结果、关键数据及其科学意义：1、荧光定量PCR检测miRNA-19b在CD患者中表达，发现活动期及复发CD患者外周血miRNA-19b升高，缓解期患者较活动期减低；活动期CD病变回肠组织分离的淋巴细胞中miR-19b高表达，病变肠黏膜组织中低表达。提示miR-19b参与了CD患者疾病活动过程。.2、流式细胞结果显示活动期CD患者外周血Th17细胞增加，Treg细胞减少；荧光免疫标记结果：活动期CD患者病变肠组织IL-17A阳性的CD4细胞增加。.3、ELISA及免疫组化法检测CD患者血及组织中Th17相关细胞因子，发现CD患者血及组织中IL-17, IL-22及IL-21水平升高，提示活动期CD患者Th17相关细胞因子升高。.4、在体及离体实验，发现肠内给予miR-19b，TNBS 诱导结肠炎减轻。过表达miR-19b，肠上皮细胞SOCS3表达减低而肠上皮细胞趋化因子（MIP-3α）升高，反之，抑制miR-19b，SOCS3表达升高而MIP-3α表达减低，提示miR-19b靶向调控SOCS3的表达介导肠上皮细胞MIP-3α产生，参与肠黏膜的炎症修复。.5.体外细胞诱导分化实验显示从非致敏Th0细胞分化为Th17，miR-19b表达增加。.以上结果提示miR-19b靶向抑制SOCS3表达，影响STAT3磷酸化，参与肠道炎性损伤过程。相关研究分别发表在Inflamm Res，Scientific Reportsd等杂志，国际会议发言3人次（UEGW及APDW）。