颌骨骨髓间充质干细胞通过外泌体miR-206调控颌面部发育的机制研究

Orofacial bone marrow mesenchymal stem cells (OMSCs) derived from neural crest cells are the main cell population that forms the maxillofacial bone. Currently, most research about maxillofacial bone development is focused on the mechanism of osteogenic differentiation of OMSCs. However, bone development is a dynamic cooperation process of osteogenesis and osteoclastogenesis, and the detailed role of osteoclasts in maxillofacial bone development is still unknown. In our previous study, the number of osteoclasts was found to be significantly up-regulated in the mandible from Wnt1-cre;Gata4fl/fl mice. This result suggests that osteoclasts may participate the development of the maxillofacial bone. Based on affymetrix miRNA array data and in vitro results, we further demonstrated that exosomal miR-206 transferred to osteoclasts could remarkably regulate osteoclast function. In summary, we will use the conditional knockout mouse and co-culture model, and analyze the communication process between OMSCs and osteoclasts, which systematically exploring the role of OMSCs in the regulation of osteoclast function. This study may provide direct insight into the causes behind the developmental malformation of maxillofacial bone, and offer potential therapeutic target to prevent maxillofacial malformation and promote the bone regeneration.

由神经嵴细胞衍生的颌骨骨髓间充质干细胞（OMSCs），是形成颌面部骨骼的主要细胞群。当前对颌骨发育的研究主要聚焦在OMSCs成骨分化调节，但骨发育是成骨和破骨动态协作的过程，破骨细胞在颌骨发育过程中的相关调控机制尚不清楚。课题组前期研究发现：在GATA4神经嵴细胞条件性敲基因小鼠颌骨中，破骨细胞数量显著上调，提示破骨细胞可能参与颌骨发育过程。结合外泌体芯片及细胞干扰等实验，课题组进一步证实OMSCs可能通过外泌体miR-206调控破骨细胞的功能。本项目拟从OMSCs和破骨细胞间对话的角度，结合条件性基因敲除小鼠和细胞共培养模型，在动物、细胞和分子水平，系统探讨OMSCs通过外泌体调控破骨细胞功能的分子机制。本课题的实施有望进一步阐明颌面发育的机理，并为颌面畸形的预防和治疗提供理论和实验基础。

颌面部骨发育过程如果受到影响将会造成颅颌面畸形。成骨细胞介导骨形成而破骨细胞则介导骨吸收，两者之间的平衡对于维持颌面部骨稳态至关重要。尽管颌骨骨髓间充质干细胞（OMSCs）在颌面部骨发育中的作用机制近年来已被初步探索，但OMSCs与破骨细胞在颌骨发育中的相互联系仍不清楚。.方法：本研究通过神经嵴细胞特异性敲除小鼠模型来研究GATA4对颌面骨发育的影响。在体外层面，首先验证了OMSCs来源的外泌体（OMExos）对破骨细胞生成和骨吸收能力的影响。从OMExos中提取miRNA，并使用Affymetrix miRNA芯片检测GATA4敲低后miRNA丰度的差异。双荧光实验验证了OMSCs中GATA4和miR-206-3p的结合，并分别在OMSCs和破骨细胞中明确了miR-206-3p的下游靶基因。最后通过体内、外实验再次验证了GATA4-miR-206-3p轴在OMSC成骨分化和破骨细胞形成中的作用机制。.结果：Wnt1 Cre;Gata4fl/fl小鼠（cKO）不仅表现出骨形成抑制，而且表现出活跃的骨吸收。将OMSCs与破骨细胞共培养，cKO组破骨细胞数目增多，而且是外泌体依赖性的。Affymetrix miRNA测序结果表明，shGATA4 OMSCs外泌体中的miR-206-3p表达下调。此外，miR-206-3p的转录活性直接受OMSCs中GATA4的转录调控。进一步证明，miR-206-3p通过直接靶向Bmp3和NFATc1在颌面部骨发育的调控中发挥了关键作用。体内拯救实验表明，局部注射OMExos和agomiR-2063p，增加了小鼠颌骨骨小梁数目、抑制破骨细胞形成，进而促进cKO小鼠的颌骨骨密度。.结论：本研究证实miR-206-3p是GATA4的一个重要下游因子，调节OMSCs和破骨细胞的功能。研究表明OMExos和microRNA agomirs可以促进骨再生，是未来治疗颌面部骨畸形的有效工具。