FoxO1-Cbfa1 轴及其上游信号通路在正畸应力诱导大鼠骨髓基质干细胞骨向分化中的作用及调控机理

The mechanism of mechanical signal trigger the biological signal to remodel the alveolar bone and osteogenic differentiation of bone mesenchymal stem cells (BMSCs) under mechanical stress is the focus of interdisciplinary studies in the fields of both orthodontics and biomechanics. The works of National Natural Science Foundation of China-Youth project has proven the vital role of Cbfa1 in mechanotransduction, however, the mechanism of its upstream regulation remains unclear. We screened some key proteins involving in bone modeling under the stress through the protein chip and gene chip technology, With a review of the literature and previous studies, we have proven the relationship between FoxO1 and Cbfa1, moreover, found the fact that FoxO1 combine the promoter of Cbfa1 directly in virtue of methods of bioinformatics. So, we hypothesize that FoxO1 might regulate the expression of Cbfa1 at the level of transcription, the axis of FoxO1- Cbfa1 might, as a key factor, regulate the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) under mechanical stress through three pertinent upstream pathways. This present project will identify that FoxO1 could combine the promoter of Cbfa1 directly by Luciferase reporter system and Chromatin immunoprecipitation and the silence and increase of FoxO1 with by means of RNAi and gene cloning techniques, furthermore, we will prove this hypothesis in vivo. Altogether, we intend to study the biological behaviour of osteogenic differentiation of bone mesenchymal stem cells (BMSCs) under stress by regulating the axis of FoxO1- Cbfa1, which will exploit the new area in promoting the local alveolar bone remodeling.

力学信号是启动牙槽骨改建最重要的生物信号之一，应力作用下骨髓基质干细胞（BMSCs）骨向分化的调控机理已成为正畸学和生物力学的交叉研究热点。本课题组前期研究证实Cbfa1在力传导中起关键作用，而其上游调控机制亟待阐明。由于新揭示的与早期骨向分化密切相关的的转录因子FoxO1与Cbfa1有较强的相关性，生物信息学也表明FoxO1可以与Cbfa1的启动子直接结合。因此，提出假设：FoxO1可能通过在转录水平调控Cbfa1的表达，即FoxO1-Cbfa1轴可能在应力调控BMSCs骨向分化中起重要作用。本项目拟通过荧光素酶报告系统及染色质免疫共沉淀鉴定FoxO1与Cbfa1启动子的直接结合，RNA干扰阻断或基因克隆上调FoxO1，激活或阻断上游通路，并进行体内实验。多层次研究应力刺激下BMSCs骨向分化的生物学行为，为通过调控该基因促进局部骨改建、缩短正畸疗程提供实验依据，开拓新的力学生物学思路。

机械刺激下局部牙槽骨组织的改建是正畸牙移动的生物学基础。应力作用下骨髓基质干细胞（bone marrow stromal cells，BMSCs）成骨向分化的转归是骨组织新生的细胞学基础。成骨细胞特异性转录因子Cbfa1作为应力介导下BMSCs骨向分化的关键基因已经明确，然而Cbfa1是通过何种途径参与调控上述过程一直未完全阐明。近年来的研究发现，FoxOs与骨组织的生成和改建密切相关。大鼠全基因组芯片检测也发现FoxOs家族成员之一FoxO1分子在应力加载后表达明显增强。.故本课题通过构建体内外应力加载模型证实：① 体外张应力作用可以激活FoxOs家族成员FoxO1/3分子的表达，且与Runx2存在明显的核内共定位。② 转录因子FoxO3可参与调控该过程中成骨功能相关基因及蛋白的表达，在应力介导骨向分化中发挥正调控作用。其可能直接通过Runx2分子对ALP进行转录激活而实现的。而FoxO1分子可能不参与上述过程的调控。③ PI3K/Akt通路可能通过调控FoxOs分子参与应力介导下BMSCs骨向分化过程。④ 大鼠牙移动模型证实正畸牙移动可导致牙槽骨骨改建增强，而在上述过程中FoxO1在成骨细胞与破骨细胞均有表达。从细胞力学、组织生物力学领域阐明了FoxOs在应力介导的BMSCs骨向分化中的作用及其可能调控机制，揭示了机械力－FoxOs－BMSCs成骨分化三者的关系，完善了正畸理论及相关骨生物力学理论。.课题已顺利完成考核目标：①由本基金所资助的直接相关文章已发表15篇（其中SCI收录论文已发表5篇，中文核心期刊10篇），已录用3篇。②会议发言：国内会议分会发言4次，壁报展示3次。③已培养博士研究生1名，硕士研究生3名。