无定型磷酸钙前驱体通过线粒体自噬参与干细胞成骨分化的机制研究

The durability and esthetics of artificial denture rely on sufficient alveolar bone support. However, bone defect can be found frequently in the region of missing tooth. Using bone augmentation technique to guide bone regeneration is the major solution for bone defect prior to prosthesis restoration. Thus, understanding the mechanism of bone formation well is of great importance. As reported recently, biomineralization of bone is initiated via amorphous calcium phosphate (ACP) precursors that were produced intracellularly and released by a single membrane vesicle to the extracellular matrix through exocytosis. Furthermore, ACP was found within mitochondria in our previous study, which has also been reported by others. Nevertheless, an unsolved enigma in this biomineralization process is how these ACP precursors are transported from mitochondrias to single membrane vesicles intracellularly. Based on systematic review and our pilot study, we propose that ACP precursors are formed within mitochondrias and transported via mitophagy intracellularly, and mitophagy regulates BMP/Smad signaling pathway by influencing Smurf1 to control bone mineralization. In order to test this hypothesis, two kinds of stem cells and rats will be used in the present study. Combined with mitochondria isolation, fluorescence double labelling in live cell, transmission electron microscope followed by three dimensional reconstruction and other techniques, we will perform the study at three intervention levels of chemicals regents, gene silencing/over-expression, specific gene knock-out to analyze the biophysical process of organelles. Also, molecular biological techniques will be used to investigate the underlying mechanism. The results of the present study will reveal a new function of mitophagy in transferring ACP intracellularly and the potential signaling pathway, which may provide help to regulate bone formation or regeneration in clinical practice.

义齿修复依赖于良好的骨支持,但临床中却常面临缺牙区骨缺损的困扰。深入了解成骨机制，促进缺损区骨形成对保障义齿修复效果至关重要。新近研究指出，钙、磷是以无定型磷酸钙（ACP）前驱体的形式,由细胞内囊泡经胞吐释放到细胞外基质来启动成骨的。此外，有研究在线粒体内也发现了ACP，但ACP如何从线粒体传递至囊泡，及其影响成骨的分子过程仍是未解之谜。基于文献调研和预实验结果，我们大胆提出：ACP可通过线粒体自噬的方式在细胞内传递，并通过Smurf1调控BMP/Smad信号通路来影响成骨。本项目以两种干细胞和大鼠为研究模型，在化学干扰、基因沉默/过表达、基因敲除三个层面进行研究，采用线粒体分离提取、活细胞荧光双标、透射电镜及三维重建等技术解析细胞器的生理过程，并通过分子生物学手段将直观表象和潜在机制结合起来，研究线粒体自噬在细胞内传递矿化前驱体ACP的新功能及其影响成骨的机制，指导成骨相关的临床诊疗。

义齿修复依赖于良好的骨支持，但临床中却常面临缺牙区骨缺损的困扰。深入了解成骨机制，促进缺损区骨形成对保障义齿修复效果至关重要。研究指出，钙、磷是以无定型磷酸钙（ACP）前驱体的形式,由细胞内囊泡经胞吐释放到细胞外基质来启动成骨的。此外，有研究在线粒体内也发现了ACP，但ACP如何从线粒体传递至囊泡，及其影响成骨的分子过程仍是未解之谜。.本项目聚焦成骨矿化前驱体ACP在细胞内的形成传递过程，以线粒体自噬为切入点，重点揭示线粒体自噬在细胞内传递ACP新的生物学功能，深入研究线粒体自噬影响干细胞成骨分化的分子机制，主要研究内容包括：1）通过干细胞成骨分化中对ACP前驱体与线粒体等细胞器的观察和分析来明确成骨分化过程中ACP前驱体是否以线粒体自噬的方式在细胞内传递。2）通过改变线粒体自噬水平来探讨线粒体自噬与成骨矿化的关系。3）干扰线粒体自噬与成骨信号通路关键蛋白的表达，研究线粒体自噬是否通过Smurf1调控BMP/Smad来影响成骨。.通过本项目的开展，取得的主要研究结果包括：1）明确了在干细胞成骨分化过程中，ACP前驱体可在线粒体内形成，并可在多种细胞内囊泡和细胞外观察到，在国际上首次报道ACP可以线粒体自噬的方式进行胞内传递。2）发现在一定范围内，增强线粒体自噬可以促进成骨矿化；抑制线粒体自噬，成果矿化效果也会受到抑制。3）明确了线粒体自噬在成骨矿化中的重要作用，指出线粒体自噬是通过Smurf1调控BMP/Smad信号通路，进而影响成骨效果的分子机制。.通过本项目的实施，解答了ACP矿化前驱体如何在细胞内进行传递的问题，加深了对骨形成过程的认识，未来可以基于该机制进行仿生，模拟线粒体稳定及传递ACP的方法设计负载ACP的促成骨材料，为加强临床骨形成效果提供新的治疗思路，促进义齿修复取得良好的功能和长期稳定的美观效果。