红景天苷调控内皮祖细胞外泌体CD73改善Perthes病中BMSC 干性丢失和分化失衡的作用机制

The initiating pathological stage of Perthes disease is decreased proliferation rate and abnormal differentiation of Bone Marrow Stromal Cells (BMSC) under hypoxic microenvironment caused by imbalance of endothelial progenitor cells and impaired vascular endothelial function. Our previous study found that CD73 from exosome secreted by endothelial progenitor cells promotes the proliferation and osteogenic differentiation of BMSC in Perthes disease. Our further research showed that the expression of AKT, mTOR, β-catenin and Oct-4 in BMSC increased, while the expressions of GSK-3β and p16 were down-regulated in BMSC, which might be related to the activation of AKT and Wnt signaling pathway. Salidroside has the effect of anti-hypoxia, angiogenesis promotion, endothelial progenitor cells activation as well as exosomes, and bone regeneration. We had confirmed that salidroside is effective during the treatment of Perthes disease in early pre-experiments. We hypothesized that salidroside can upregulate EPCs to promote the synthesis of CD73 and release exosomes contained CD73, which further activates AKT / mTOR and Wnt / β-catenin signaling pathway to promote the proliferation and osteogenic differentiation of BMSCs. In this study, we intend to validate the hypotheses from the animal-cell-molecular level, and to analyze the pathways and targets of th key molecules regulation of downstream effects. The result of this study will provide new ideas and new drugs for the prevention and treatment of Perthes disease.

儿童股骨头缺血坏死(Perthes病)的始动病理环节为内皮祖细胞失调、血管内皮功能受损，导致局部乏氧微环境下骨髓基质干细胞干性丢失和分化失衡。我们研究发现：内皮祖细胞来源的外泌体CD73能促进Perthes病BMSC的干性增殖和成骨分化。进一步研究发现BMSC中AKT、mTOR、β-catenin以及Oct-4的表达增高，而GSK-3β和p16表达下降，推测可能与AKT和Wnt信号通路的激活有关，具体机制不明。活血化瘀中药红景天苷促血管新生，可活化内皮祖细胞外泌体，促进骨再生，预实验已证实其治疗Perthes病有效。我们假设：红景天苷活化内皮祖细胞促进CD73的合成及通过外泌体释放，进而激活AKT/mTOR和Wnt/β-catenin信号通路促进BMSC的干性增殖和成骨分化。本项目拟从动物-细胞-分子层次验证假说，并解析药物调控下游效应的途径及靶点。此研究将为该病的防治提供新思路和新药物。

儿童股骨头缺血坏死(Perthes病)的始动病理环节为内皮祖细胞失调、血管内皮功能受损，导致局部乏氧微环境下骨髓基质干细胞干性丢失和分化失衡。前期发现内皮祖细胞来源的外泌体CD73能促进Perthes病BMSC的干性增殖和成骨分化，推测可能与AKT和Wnt信号通路的激活有关。活血化瘀中药红景天苷促血管新生，可活化内皮祖细胞外泌体，促进骨再生。本项目主要研究内容包括：1.内皮祖细胞外泌体CD73对Perthes病BMSC干性增殖和分化方向的影响：2.内皮祖细胞外泌体CD73对BMSC中AKT/mTOR和Wnt/β-Catenin信号通路调控机制：3.红景天苷调控内皮祖细胞外泌体CD73的信号通路以及红景天苷治疗Perthes病的作用机制：4.结合体外组织、细胞和体内动物实验，根据上下游通路和靶分子分析结果，阐明红景天苷通过HIF-1α通路对内皮祖细胞活性和功能以及分泌CD73再通过相关信号通路对BMSC的干性增殖和分化方向的调控，红景天苷通过调控CD73治疗Perthes病。已经取得的结果包括：1.脐带血内皮祖细胞来源的外泌体，注射至Perthes病兔模型体内，结果发现能有效改善早期Perthes病，治疗组动物的股骨头骨密度和新生骨质明显增加。进一步分析内皮祖细胞外泌体中活性成分，发现CD73显著促进BMSC的干性增殖和成骨分化，在治疗兔早期Perthes病实验中具有重要作用，在加入CD73特异性阻断剂APCP干预后再次评估发现，该内皮祖细胞外泌体对Perthes病的促进骨新生作用大大降低。CD73的作用机制进一步研究发现Perthes病兔BMSC中AKT和Wnt信号通路中的关键蛋白AKT、mTOR、β-catenin以及与干性增殖有关的基因Oct-4、Sox-2的水平明显增高，而GSK-3β和衰老基因p16,p21表达水平明显下降。利用脐带血内皮祖细胞来源的外泌体，注射至Perthes病兔模型体内，结果发现能有效改善早期Perthes病，治疗组动物的股骨头骨密度和骨小梁数量明显升高。进一步的实验发现红景天苷可以通过HIF-1α信号通路促进内皮祖细胞中CD73的合成并促进外泌体中CD73的释放。和细胞实验也验证了红景天苷对早期Perthes病的治疗作用。本研究将为该病的防治提供新思路和新药物。