Nell-1基因在牵张成骨过程中的作用及其机制研究

While the surgical procedure of distraction osteogenesis (DO) has been successfully used for a variety of craniofacial deformities, the precise molecular mechanism is still unclear. Nel-like protein 1 (Nell-1) is a novel osteoinductive molecule that has been found to promote osteoblast cell differentiation and mineralization. Canonical Wnt/β-catenin signal pathway is one of the most important regulatory systems in bone metabolism and response of osteoblasts to mechanical stress. Nevertheless, knowledge about their expression in distraction osteogenesis remains limited. We raise the hypothesis that Nell-1 may play an important role in the formation of the regenerated bone during DO. Nell-1 might promote the regenerated bone formation, and this effect might correlate with Wnt/β-catenin signal pathway. The aim of this study is to evaluate the potential roles and regulatory mechanism of Nell-1 in DO. The expression of Nell-1, Wnt1, Wnt3a and β-catenin will be investigated in vitro with osteoblasts cultured on a distraction device with or without the application of Wnt/β-catenin pathway inhibitor DKK-1 or, and in vivo with a rabbit mandibular DO model. The results of this study might indicate that Nell-1 and Wnt/β-catenin signal pathway might be potential therapeutic targets in accelerating bone regeneration during DO and might be helpful to elucidate the molecular mechanism of DO.

牵张成骨技术已被成功应用于矫治多种颅颌面畸形，但其确切的分子机制尚不十分清楚。Nell-1是一种能够促进成骨细胞成熟和分化的成骨因子，而Wnt/β-catenin信号转导通路参与了成骨细胞对机械应力的响应过程。目前未见有关于Nell-1在牵张成骨过程中的作用及其调节机制的研究。本项目提出以下假设：Nell-1参与了牵张成骨新骨生成的过程，并可以促进新骨生成，其作用机制可能与Wnt/β-catenin信号转导通路有关。本项目拟采用成骨细胞体外牵张模型，分别在给予和不给予Wnt/β-catenin信号通路阻断剂DKK-1的情况下，检测Nell-1、Wnt1、Wnt3a及β-catenin等的表达情况，并采用兔下颌骨牵张成骨模型检测上述基因的表达，旨在探讨Nell-1基因在牵张成骨过程中的作用及调节机制，有助于进一步阐明牵张成骨的分子机制，有望为应用基因工程技术促进牵张成骨新骨生成奠定实验基础。

研究背景：牵张成骨是目前口腔颌面外科领域一种重要的临床技术，但其分子学机制目前尚不清楚，还有待进一步研究。Nell-1基因是一种密切参与了机体成骨过程的基因。而Wnt/β-catenin信号转导通路参与了成骨细胞对机械应力的响应过程。目前未见有关于Nell-1在牵张成骨过程中的作用及其调节机制的研究。本项目提出以下假设：Nell-1参与了成骨细胞对牵张应力应答的生物学过程，并可以促进成骨细胞分化，其作用机制可能与Wnt/β-catenin信号转导通路有关。.研究目的：研究不同牵张应力及作用时间对大鼠成骨细胞Nell-1蛋白表达的影响。.方法：本研究对体外培养的小鼠成骨细胞分别施加0，2000，20000和100000microstrain大小牵张应力，于牵张后4h、12h采用Western blot法研究Nell-1蛋白表达随牵张力大小及作用时间的不同而发生的改变，采用Western blot法检测骨钙蛋白（osteocalcin，Ocn）蛋白表达水平的改变，采用PNPP法检测碱性磷酸酶（alkaline phosphatase, ALPase）的比活性。.结果：在生理性牵张应力作用下，Nell-1蛋白、Ocn蛋白、ALP的表达随牵张力的增大而增加。在病理性牵张应力作用下，三者的表达则显著降低。.结论：Nell-1蛋白参与了成骨细胞将应力刺激信号转化为生物学信号的过程，可能密切参与了机体牵张成骨的过程。Nell-1基因表达与Wnt/β-catenin信号通路的关系尚需进一步研究。