IL-17A诱导破骨细胞分化及调控Notch2/RBP-J信号转导的机制研究

One of the risk factors for bone loss in periodontal disease is the increase of bone-resorbing osteoclasts. Both IL-17A and Notch signaling play important roles in osteoclastogenesis and bone resorption. However, the underlying mechanism between the interaction of IL-17A and Notch/RBP-J signaling pathway is not well-defined. Our pilot study demonstrated that IL-17A augmented NF-κB transcriptional activity and Notch2 expression level. Meanwhile, IL-17A down-regulated RBP-J expression level at early stage while up-regulated RBP-J expression level at later stage. Potential NF-κB binding sites are predicted on the Notch2 and RBP-J promoter regions. These results suggested the potential effect of IL-17A/NF-κB on Notch2/RBP-J. The aim of these studies is to investigate which member of NF-κB family regulates Notch2/RBP-J in IL-17A-induced osteoclastogenesis and bone resorption．Furthermore, the NF-κB binding sites in Notch2 and RBP-J promoter regions will be explored. The results obtained from these studies will provide useful information for inhibition of inflammatory bone destruction related to periodontal disease.

破骨细胞过度活跃是引发炎症性牙槽骨吸收的病理基础。IL-17A和Notch信号在破骨细胞分化及骨吸收中发挥重要作用。 但IL-17A与Notch/RBP-J信号通路间的相互关系尚不清楚。本课题组前期研究表明：IL-17A具有诱导NF-κB转录活性及促Notch2/RBP-J信号转导的生物学功能；软件分析Notch2和RBP-J启动子均存在NF-κB的结合位点。由此推测IL-17A/NF-κB对Notch2/RBP-J信号通路的调控作用可能影响破骨细胞分化和骨吸收过程。因此，本课题拟进一步探讨IL-17A诱导破骨细胞分化及炎症性牙槽骨吸收过程中，参与调控Notch2/RBP-J信号通路的NF-κB家族特定成员，明确受NF-κB信号调控的Notch2和RBP-J启动子区域具体位点。深入探讨IL-17A/NF-κB调控Notch2/RBP-J信号的分子机制，可为抑制炎症性牙槽骨吸收提供新思路。

本研究采取细胞培养、实时定量PCR、蛋白印迹和荧光素酶报告基因等分子生物学手段和基因敲除动物模型，观察炎症因子诱导破骨细胞分化和软骨细胞去分化的相关指标，探讨Notch/RBP-J信号在破骨细胞分化中的作用，并探讨破骨细胞分化环状RNA分子的表达情况。研究发现：（1）活化Notch2信号促进破骨细胞分化，并下调转录因子RBP-J的转录活性；阻断Notch2信号抑制破骨细胞分化，升高转录因子RBP-J的蛋白表达水平，并上调转录因子RBP-J的转录活性；因此，Notch2信号正向调控破骨细胞分化过程和反向调控转录因子RBP-J活性。（2）IL-17A诱导破骨细胞分化过程中RBP-J与P65两核转录因子存在相互作用。(3)在破骨细胞分化过程中，环状RNA分子整体表达水平下降，并初步发现一新环状RNA分子circEMR1。（4）通过TetraOne打靶技术成功构建Rbpj基因敲除小鼠动物模型。（5）炎性因子诱导软骨细胞去分化过程中，整体DNA 甲基化水平增高，DNMT3A 为参与调控软骨细胞去分化的关键DNA甲基化转移酶。上述结果表明RBP-J转录因子和环状RNA分子可能成为调控破骨细胞分化和骨吸收的基因靶点；破骨细胞和软骨细胞相互作用参与调控软骨和骨基质代谢。