应力下microRNA-26a-5p靶向调控Wnt5a对下颌骨牵张成骨中新骨形成影响的分子作用机制

The aim of this project is to promote the new bone formation in mandibular distraction osteogenesis(DO), and the key research point in this study is to investigate the mechanism of microRNA-26a-5p(miR-26a-5p) as mechanical signal transmission molecular in DO. Based on our previous works, the animal models of DO is established, and we transfect Lv-miR-26a-5p、Lv-miR-NC、Lv-ASO-26a-5p or Lv-ASO-NC into BMSCs respectively in vitro, and then we deliver these BMSCs into distraction gap respectively. After down-regulated or up-regulated the expression of miR-26a-5p,we investigate the expressions of Wnt5a、 representative molecules of non-canonical Wnt and osteogenic genes during distraction osteogenesis, and we want to understand the definite mechanical signal transmission mechanisms of miR-26a-5p in distraction osteogenesis through above works. In addition, in this study, a cellular mechanical stimulation apparatus which could simulate biomechanic environment of DO is established. The miR-26a-5p transfected BMSCs are loaded respectively by tension in this cellular mechanical stimulation apparatus , and the miR-26a-5p、Wnt5a and its downstream genes are investigated too, we hope that the results of the cell biomechanic test could verify that of animal experiment. In general, in this project, all the above works help us to understand the effects on new bone formation and the roles in mechanical signal transmission of miR-26a-5p targeting Wnt5a during distraction osteogenesis process.

本课题以促进下颌骨牵张成骨（DO）新骨形成为研究目标，以明确microRNA-26a-5p (miR-26a-5p)在牵张成骨中的力学信号作用机制为研究核心，建立下颌骨牵张成骨动物模型，将调节miR-26a-5p功能的慢病毒载体转染入 BMSCs后分别导入牵张区，研究上调和下调miR-26a-5p后Wnt5a、非经典Wnt途径关键分子及成骨特异基因在DO成骨进程中的表达变化，从而明确miR-26a-5p对新骨形成的影响及其参与成骨的力学转导模式。此外，本研究建立了体外模拟DO应力环境的细胞力学刺激系统，将转染miR-26a-5p后的BMSCs置于该系统中进行张应力刺激，探讨miR-26a-5p在外施加力学刺激中对BMSCs骨向分化的分子调控机制，研究其力学信号传导作用机制，并对上述动物实验的结果进行验证，同时为深入认识miR-26a-5p在牵张成骨治疗中的促成骨作用奠定细胞力学实验基础。

本项目以促进下颌骨牵张成骨新骨形成为研究目标，以明确miR-26a-5p(microRNA-26a-5p)在牵张成骨中的力学信号机制为核心开展了研究工作。项目进展顺利，圆满完成了计划书的任务要求。主要研究内容和成果汇报如下： .①我们证实miR-26a-5p负向调控间充质干细胞的骨向分化。.②我们证实miR-26a-5p与Wnt5a 3’UTR存在直接靶向关系。.③我们证实miR-26a-5p抑制了Wnt5a对Wnt非经典信号通路的激活，导致间充质干细胞骨向分化被抑制。miR-26a-5p抑制Wnt/Ca2+信号通路并上调了Wnt经典信号通路β-catenin表达，表明在miR-26a-5p参与调控的干细胞骨向分化过程中Wnt经典信号通路和Wnt非经典信号通路之间存在着密切的联系。.④我们证实上调miR-26a-5p会抑制牵张成骨动物模型中新骨形成，降低成骨速率，而下调miR-26a-5p将显著提高牵张成骨新骨的质量和数量，这是促进新骨形成，提高治疗效果的有效新手段。.⑤我们实现了牵张成骨下颌骨三维有限元建模，对关节区和骨延长区进行了应力计算，延长区应力分布揭示了骨生成速率和成骨质量不同，其中ASO-miRNA-26a-5p组成骨较好，关节区及延长区不良应力产生较少。 .⑥我们证实Lv-miR-26a-5p及Lv-ASO-26a-5p具有稳定调控干细胞miR-26a-5p表达的能力。体外细胞力学试验证实miR-26a-5p是张应力下间充质干细胞骨向分化的关键信号分子，miR-26a-5p过表达可显著抑制其成骨分化，抑制miR-26a-5p表达可显著促进其骨向分化。.⑦我们证实张应力作用下miR-26a-5p通过Wnt/Ca2+信号通路参与了间充质干细胞骨向分化的调控，miR-26a-5p-Wnt/Ca2+是关键的应力转导途径，张应力刺激下调了miR-26a-5p的表达，增强Wnt5a的表达，最终促进了间充质干细胞骨向分化的力学响应。. 本项目建立了下颌骨牵张成骨动物模型及体外细胞力学刺激系统，体内外结合研究了miR-26a-5p在应力下对间充质干细胞骨向分化的分子调控作用。项目首次揭示了牵张成骨中Wnt5a-miR-26a-5p通路调节骨发生发育的机制，明确了张应力下miR-26a-5p对新骨形成的影响及力学信号转导分子作用机制，具有重要的科学意义和学术价值。