胞外力学信号偶联调控间充质干细胞成软骨分化的机制研究

A key challenge in cartilage tissue engineering is to learn how to direct the differentiation of stem cells toward specific fates and maintain their phenotypes. The role of mechanical signals in the regulation of stem cell differentiation has been widely and deeply studied, and provides a new approach to the solution of this problem. We have devoted to study the mechanotransduction mechanism during chondrogenic differentiation of MSCs (mesenchymal stem cells), and found that applied force could induce intrinsic TGF-beta to promote the chondrogenic differentiation of MSCs; however, it failed to keep the chondrogenic phenotypes stable. Recently, a new factor was discovered to regulate stem cell fate: the stiffness of the substrate. It was reported in Cell in 2006 that such regulation was mediated by transduction of intrinsic forces, which evidenced the existence of a new mechanotransduction pathway. In 2012, Nature commented that these two forces are coupled to determine stem cell fate. The pilot studies of our research group found that the substrate stiffness had significant regulatory effects on the chondrogenic differentiation of MSCs. However, it is not clear that if extrinsic forces have coupled effects with intrinsic forces. Based on our previous studies in extrinsic mechanotransduction pathway, this project is aiming to establish models of intrinsic force-induced chondrogenic differentiation and study the signal coupling in the mechanotransduction of intrinsic forces. We also plan to find the common signal molecules shared by these two forces and to provide a target for extrinsic-intrinsic force coupling. Moreover, we are trying to explore the coupled effects and mechanism of mechanotransduction of these two kinds of forces. This project will not only lay solid foundation to optimize the mechanism of mechanotransduction, but also promote the application of biomechanics to cartilage tissue engineering.

促进干细胞定向成软骨分化并维持表型稳定是软骨组织工程研究的热点和难点问题。力学信号对干细胞分化调控研究的深入和进展，为这一问题的解决提供了新方向。课题组发现外源性应力可激活内源TGF-β来促进间充质干细胞（MSCs）成软骨分化, 但表型稳定性欠佳。2006年Cell首次报道基质硬度可启动内源性力学转导途径来调控MSCs的分化方向，并证实该途径对分化表型的维持相对稳定。2012年Nature报道内、外源力学信号在调控MSCs分化时存在偶联。本课题预实验发现硬度刺激可诱导MSCs成软骨分化，以此为基础研究内、外源性应力刺激对MSCs成软骨分化的偶联调控，具有高度创新性和可行性。本项目拟建立MSCs成软骨分化的硬度-内源性应力刺激模型，研究内源性力学转导过程中的信号偶联及其与外源性力学转导途径的联系；建立MSCs成软骨分化的内、外源应力共刺激模型，研究其偶联效应并拓展在软骨再生及修复中的应用。

力学信号对干细胞分化调控研究的深入和进展，为解决软骨组织工程中干细胞定向成软骨分化并维持表型稳定提供了新的思路。目前比较清楚内源性和外源性应力均可调控成软骨分化，但内外源性应力共调节成软骨分化的机制仍不清楚。本课题主要研究内容与结果如下：.1.采用不同硬度聚丙烯酰胺基质硬度材料（PAAm）以及Cytosoft®材料，成功建立成软骨分化的硬度-内源性应力刺激模型，采用PCR、免疫荧光、HE染色等技术，研究发现内源性应力通过调节细胞骨架以及细胞形态调节细胞的分化方向；YAP/TAZ信号是不同硬度刺激调控成软骨分化过程中重要的调控因子，且其调控机制与F-actin细胞骨架以及细胞形态有关。表明硬度刺激向胞内传递过程中，Wnt-Hippo信号以及细胞骨架系统存在交互效应。.2.研究了外源性应力对MSCs成软骨分化调控效应，发现：MAPK/ERK对成软骨存在负调控效应，ERK表达在成软骨诱导培养早期一过性升高并在后期逐渐降低，但在培养后期进行外源性应力加载会使其再次升高。提示MAPK/ERK信号正是外源应力刺激调控成软骨分化的过程中的“缓冲阀”。.3.采用基因芯片技术，研究内源性应力刺激调控MSCs成软骨分化的基因差异表达谱，通过GO分析和KEGG分析，提示未来可研究GnRH信号通路、粘附斑、Rap1信号通路、ERBB信号通路、PKC等信号在内源性应力调控干细胞分化中的机制。.4.建立了内、外源性力学信号共刺激模型，分别通过体外研究、体内研究，研究内、外源力学偶联信号对间充质细胞成软骨细胞分化的调控效应，明确了内、外源性力学信号转导对MSCs成软骨分化的调节具有偶联效应。.本项目成功建立了成软骨分化的内、外源性应力刺激模型，明确了内、外源性力学信号转导对成软骨分化的调控既有交叉又有不同；建立了内、外源应力共刺激模型，发现二者具有偶联效应，未来可应用于软骨再生及修复应用。.