成骨细胞分化过程中线粒体能量代谢调控及机制研究

As an aging-induced skeletal degenerative disease, osteoporosis is tightly associated with decreasing osteoblast differentiation. Mitochondria serve as the source of cellular energy and are closely related to osteoblast differentiation. In recent years, the PINK1/Parkin-mediated mitophagy which initiated from phosphorylated ubiquitin has been demonstrated to play an important role in the maintenance of mitochondrial function. However, whether this pathway is associated with the decreasing osteoblast differentiation during osteoporosis and the related mechanisms remain still unclear. In order to explore the intrinsic relationship between mitochondrial energy metabolism and osteoblast differentiation, this project intends to perform the following investigations: 1) To observe the difference of the PINK1 experssion in osteoblasts between osteoporotic mice and normal ones; 2) To explore the osteoblast differentiation and related molecular mechanisms aided by the techniques of knockout or up-regulation of PINK1; 3) To synthesize and screen the phosphorylated polyubiquitins capable of activating the PINK1/Parkin mitophagy in osteoblasts; 4) To detect the therapeutic effect of bone-targeted phosphorylated polyubiquitins on osteoporosis animals. The results not only can reveal novel mechanisms and targets for the regulation of osteoblast differentiation, but also provide new intervention strategy for the diseases related to osteoblast differentiation.

骨质疏松是骨骼衰老的表现，其发生与成骨细胞分化能力的降低密不可分。线粒体作为细胞能量供应的主要来源，其功能维持与成骨细胞分化密切相关。磷酸化泛素参与启动的PINK1/Parkin线粒体自噬通路在线粒体功能的维持方面发挥重要功能，但该通路与骨质疏松发展过程中成骨细胞分化能力的降低是否存在关联及相关分子机制尚不清楚。为了探索线粒体能量代谢与成骨细胞分化的内在关联，本项目将开展如下研究：1)考察骨质疏松小鼠和正常小鼠骨组织中成骨细胞的PINK1水平差异；2)通过定点敲除或上调PINK1水平研究成骨细胞分化能力的改变及相关分子机制；3)筛选并制备能够促进成骨细胞成骨分化的PINK1/Parkin自噬通路磷酸化泛素分子；4）检测骨靶向修饰的磷酸化泛素分子对骨质疏松动物模型的治疗效果。预期将发现成骨细胞分化调控的新机制和新靶点，为骨质疏松症治疗提供全新的干预手段。

成骨细胞是参与骨代谢的一种重要骨细胞，成骨分化是一高能量代谢过程，需要大量完整和活跃的线粒体参与。在骨代谢紊乱疾病（骨溶解、骨质疏松、骨关节炎等）中均存在骨细胞的线粒体自噬异常现象。PINK1/Parkin信号通路中介导损伤线粒体泛素化，Parkin能够帮助细胞及时清除损伤线粒体，指导损伤线粒体的自噬清除。在成骨细胞分化过程中，线粒体自噬机制尚未阐明，对成骨细胞线粒体自噬机制进行研究，有助于对该过程精准调控，及时干预成骨分化异常。本课题项目在构建小鼠成骨细胞分化模型的基础上，探讨了PINK1靶点对细胞成骨分化的影响；合成对线粒体自噬起关键作用的结构明确的磷酸化的二泛素，发现远端Ub磷酸化可以有效激活野生型Parkin，仅有近端Ub磷酸化的二泛素不能激活野生型Parkin，但可激活磷酸化Parkin；合成末端酰胺化的磷酸化单泛素探针，对Parkin活性激活机制进行了针对性表征；利用成骨细胞线粒体自噬模型和多种K6型泛素表达载体，在细胞水平研究确定了K6泛素对于线粒体自噬情况的影响及在线粒体自噬过程中的重要作用，明确了泛素的K6位点在成骨细胞分化过程中的关键作用。本项目的研究为小鼠成骨细胞分化过程中有效干预及进一步明确成骨分化过程中线粒体代谢的机制提供了重要的实验数据，为机制的阐明奠定了坚实的基础。