牙周膜CREB磷酸化在正畸牙移动成骨中的作用

Teeth are moved through alveolar bone by the application of adequate orthodontic forces，but the exact mechanism of the bone formation in the tensile side is still unclear. CREB is a kind of phosphorylation-dependent transcription factors, which was proved to regulate the expression of vast number of growth factors and cytokines. Our preliminary study found that the expression and phosphorylation of CREB is specifically upregulated in the periodontal ligament cells (PDLs) of the tensile side during tooth movement. We raise a hypothesis that phosphorylation of CREB in the tensile side may regulate bone formation during orthodontic tooth movement. Based on bioinformatics methods and micro-array results, potential CREB target genes were chosen. Firstly, the expression patterns of CREB, P-CREB and potential CREB target gene in the PDLs under tensile force were detected in vivo and in vitro. Secondly, CREB was activated or inhibited by overexpression of constitutive CREB or dominant negative CREB in the PDLs. And the expression of the potential CREB target genes is detected by RT-PCR and western blot. Induction of BMSC migration and osteogenic differentiation are detected by transwell and induction medium. Furthermore, are used to predict its downstream target gene. The binding interaction between p-CREB and its target genes will be further confirmed by dual luciferase assay, EMSA and ChIP assay. This study will help to understand function of PDLs expressed CREB in bone formation during orthodontic tooth movement.

正畸牙移动牵张侧出现新骨形成的机制尚未明确。CREB是一种磷酸化依赖性的转录因子，调控多种细胞生长因子及趋化因子的表达。项目组前期研究发现，CREB磷酸化特异性发生在小鼠正畸牙移动模型牵张侧的牙周膜细胞中，同时体外模型中周期性张应力的牙周膜细胞CREB表达及磷酸化水平上调。项目组提出假设：牙周膜细胞CREB磷酸化在正畸牙移动牵张侧的成骨过程中发挥重要作用。通过生物信息学预测结合表达谱芯片结果sdf1、fgf2为CREB潜在靶基因。首先检测体内外牙周膜牵张应力条件下CREB的表达及磷酸化，检测sdf1、fgf2表达，分析CREB磷酸化与sdf1、fgf2的相关性；分别在牙周膜细胞中过表达CREB和抑制CREB后检测牙周膜细胞条件培养基对骨髓基质细胞迁移、成骨向分化的作用，检测下游潜在靶基因sdf1、fgf2表达。进一步用双荧光素酶实验、染色体免疫共沉淀实验检测CREB对靶基因结合与调控作用。

牙在正畸力作用下在牙槽骨中移动过程中,其牵张侧出现新骨形成的机制还不明确。CREB是一种磷酸化依赖性的转录因子，调控多种细胞生长因子及趋化因子的表达。课题组通过牙周膜细胞体外周期性牵张力模型mRNA芯片数据的网络分析发现，CREB是其中网络调控中多个重要中心的一员。通过进一步的验证，我们发现CREB在周期性张应力的牙周膜细胞中表达及磷酸化水平上调。为探讨张应力状态下的牙周膜细胞CREB磷酸化在正畸牵张成骨过程中的作用，我们同时建立小鼠正畸牙移动的模型，通过real-time PCR，western blot和免疫组织化学方法检测体内外牙周膜牵张应力条件下CREB表达及磷酸化上调。在体外模型中我们发现，在牙周膜细胞中过表达CREB可以使牙周膜细胞分泌促进骨髓基质干细胞迁移的细胞因子。其中通过对细胞上清的检测，我们发现FGF2和SDF1的蛋白水平升高。在小鼠模型中，我们也发现FGF2和SDF1在牙周膜牵张侧出现表达上调。 通过过表达和抑制CREB实验我们发现，CREB可促进FGF2和SDF1表达上调。CREB，作为一个转录因子，可以通过转录调控FGF2和SDF1促进他们的表达。本研究结果对揭示牙周膜细胞CREB磷酸化可促进骨髓基质干细胞的迁移，在正畸牵张成骨的调控机制有重要作用。