成骨细胞外泌体microRNA对间充质干细胞成骨分化的调控作用及机制

Accumulating evidence supports that osteoblasts promote mesenchymal stem cells (MSCs) differentiation toward osteoblasts by secretory factors, but the exact machanisms are unclear. In our previous study, for the first time, we detected the existence of miRNA component in the exosomes from osteoblast MC3T3-E1 cells. By analyzing these miRNAs function and expression pattern during osteogenic differentiation, we deduced that these exosomal miRNAs tend to promote mesenchymal stem cells (MSCs) osteogenic differentiation if they shutted into MSCs. Based on above findings, we proposed that osteoblast may induced MSCs osteogenic differentiation by exosome-mediated miRNA. In this study, we plan to validate whether exosomes from mineralizing osteoblasts could shuttled into the cytoplasms of MSCs and promote its osteogenic differentiation. Then, we will confirmed that miR-2861 is a key effector of osteoblast exosomes to induce MSCs osteogenic differentiation. Finally, we will continue to explore the possibility that miR-2861 and other osteoblast exosomal miRNAs cooperatively promote MSCs osteogenic differentation. Therefore, our study may clarify new regulatory machanisms of MSCs osteoblast differentiation, and may also help to provide new ways to control and regulate osteogenesis of MSCs and facilitate future clinical applications of MSCs in tissue engineering.

有证据表明成骨细胞能以外分泌方式促进间充质干细胞的成骨分化，但其机制尚不清楚。申请者的前期研究首次发现了成骨诱导的MC3T3-E1细胞的外泌体（exosome）中存在miRNA，通过分析miRNA的功能和表达模式，我们推断这些成骨细胞外泌体中的miRNA进入间充质干细胞后将促进其向成骨方向分化。据此，我们提出"成骨细胞可能通过其外泌体miRNA调控间充质干细胞的成骨分化"的科学假说。本课题拟进一步明确矿化期成骨细胞的外泌体能否进入间充质干细胞内并促进其成骨分化；验证miR-2861是成骨细胞外泌体促进间充质干细胞成骨分化的关键效应因子；探讨miR-2861与其他的外泌体miRNA协同作用，共同调控间充质干细胞成骨分化的可能性。研究结果将为阐明间充质干细胞成骨分化的调控机制、建立骨组织损伤修复与重建方法提供新的理论依据。

本项目主要围绕外泌体介导的成骨细胞对间充质干细胞的促成骨作用开展研究。结果发现成骨细胞分泌的外泌体释放后能够进入到间充质干细胞内，对后者的成骨分化具有明显的促进作用；这一作用与成骨细胞外泌体通过细胞通讯释放其miRNA内容物，显著改变受体间充质干细胞的miRNA表达谱有关；我们进一步发现成骨细胞外泌体作用后多个显著上调的miRNA共同抑制了Axin1基因，进而激活并促进了Wnt-β-catenin信号通路，从而促进了间充质干细胞的成骨分化。本课题的意义在于在细胞水平揭示了外泌体miRNA作为一种特殊的细胞间调控因子参与了成骨细胞与间充质干细胞的调控，这是对既往以分泌蛋白作为主要调控方式的补充。鉴于外泌体的工程化水平的不断提高，本研究结果对于进一步将外泌体应用于骨组织的损伤修复与重建奠定了理论基础。此外，我们还从外泌体中还发现了PRELP蛋白及miR-5100等一批具有促成骨作用的因子。项目相关成果共发表学术论文9篇。