自噬-外泌体途径介导的破骨前体细胞/成骨细胞间通讯在磨损颗粒诱导骨溶解中的作用及机制研究

Aseptic loosening is the most common complication of artificial joint replacement and the key factor affecting the survival time of prosthesis . Wear particles generated from prosthesis have been thought to be the main cause of aseptic loosening, yet the underlying molecular mechanism remains elusive. Our previous studies found that the functional activity of osteoblasts could be upregulated by RAW264.7 cells-derivied exosomes, suggesting that exosome-mediated intercellular interactions might play an important role in the process of wear particle-induced osteolysis. Moreover, we also found that the level of miRNAs in RAW264.7 cell-derived exosomes could be inhibited by wear particle-induced autophagy. Accordingly, we proposed the following hypothesis: the osteoclast precursor-derived exosomes could be inhibited by wear particle-induced autophagy, which might in turn promote osteoblast activity, thus resulting in the genesis of osteolysis. Based on molecular/cellular/animal level study, Our project aims to illustrate the function of autophagy-exosome pathway in crosstalk between osteoclast precursors and osteoblasts during the pathogenesis of osteolysis, and explore the possibility of autophagy and exosomes-targeted interventions. This project will provide new insights for clinical prevention and treatment of aseptic loosening.

假体无菌性松动是人工关节置换术后的常见并发症，也是影响假体使用寿命的关键因素。目前研究认为，假体产生的磨损颗粒诱导的骨溶解是引起无菌性松动的主要原因，但其中的分子机制尚不明确。申请人前期研究发现，RAW264.7细胞分泌的外泌体能够上调成骨细胞的活性，提示外泌体介导的细胞间通讯可能在磨损颗粒诱导骨溶解过程中发挥了重要作用。同时，磨损颗粒诱导的自噬能够抑制RAW264.7细胞分泌外泌体及其中miRNA的表达。据此我们提出假说：磨损颗粒可通过诱导破骨前体细胞产生自噬，从而抑制破骨前体细胞分泌外泌体及其中miRNA的表达，影响成骨细胞活性，进而参与骨溶解的发生。本课题拟从分子-细胞-动物三个层面，系统阐明磨损颗粒通过破骨前体细胞自噬-外泌体途径调控成骨细胞活性，引起骨溶解的病理过程，并探讨通过干预自噬或利用外泌体进行防治骨溶解的可能性，为治疗无菌性松动提供新的理论依据和实验基础。

磨损颗粒诱导的炎性骨溶解是引起人工关节假体无菌性松动的主要原因，也是影响假体使用寿命的关键因素。目前关于磨损颗粒导致炎性骨溶解的分子机制尚不明确。本项目通过细胞实验发现，磨损颗粒刺激后可诱导RAW264.7细胞产生自噬，抑制破骨前体细胞源性外泌体的分泌，及其中miRNA的表达。进一步miRNA测序发现，磨损颗粒刺激后差异性表达的miRNA有16种，其中miR-320-3p表达明显上调，miR-29a-3p表达明显下降。随后，我们将磨损颗粒处理前后破骨细前体细胞分泌的外泌体分别与骨髓间充质干细胞（BMSCs）共培养，发现磨损颗粒刺激后破骨细前体细胞分泌的外泌体，其诱导BMSCs成骨分化的能力显著降低。我们还构建了小鼠颅骨骨溶解模型，并发现磨损颗粒刺激后颅骨组织中的自噬标志蛋白LC3明显上升，而成骨细胞相关蛋白osteicalcin、ALP、BSP和type 1 collagen的表达明显下降，而利用自噬抑制剂和小鼠骨髓造血干细胞来源的外泌体进行干预治疗后，能明显改善成骨细胞活性，microCT扫描和甲苯胺蓝兰染色进一步证实小鼠骨髓造血干细胞来源的外泌体能显著缓解磨损颗粒能诱导的颅骨骨溶解。本项目从分子-细胞-动物三个层面，系统阐明了磨损颗粒通过破骨前体细胞自噬-外泌体途径调控成骨细胞活性，引起骨溶解的病理过程，并发现通过干预自噬或利用外泌体治疗可逆转骨溶解，为治疗无菌性松动提供新的理论依据和实验基础。