骨关节炎软骨的病理性ECM硬度调控软骨细胞表型的效应及机制研究

Osteoarthritis (OA) is one of the most often happened worldwide orthopedic disorders, current administrated drugs are mainly for inflammation inhibition or pain release. Meanwhile, besides inflammation factors, OA progression is also associated with mechanical changes of cartilage matrix. Previous studies have showed that the mechanical microenvironment can determine the fate of cells, and diseases such as tumor are associated with stiffening of extracellular matrix (ECM) as well as activation of YAP, indicating that YAP may also involved in OA pathological progress. So we hypothesize that the pathological mechanical microenvironment of articular cartilage matrix could affect the chondrocyte phenotype in OA. Our preliminary study also found stiffening of articular cartilage ECM from OA patient as well as higher nuclear localization of YAP, further supporting our hypothesis. So in this proposal, we will carry out the following studies: 1) Firstly to test the stiffness of ECM and YAP activation in OA tissue and cells in vitro; 2) To fabricate cell culture substrates with different stiffness mimicking the ECM from OA and normal cartilage and investigate its effect on chondrocyte phenotype maintenance and underlying mechanism; 3) To develop YAP conditional knockout mice to prove the effect of YAP activation on OA pathological progress；4) To controlled release verteporfin by joint injection of chitosan microsphere to see whether it will slow down OA process and its possible mechanism in animal model. The findings will help to understand the role of YAP in OA progression and its underlying mechanism, thus providing new knowledge and method for OA treatment.

骨关节炎（OA）目前治疗药物多是处理炎症或疼痛等对症治疗，而OA的启动除炎症等因素，软骨基质力学有病理性改变。以往研究表明力学微环境影响细胞命运，肿瘤等疾病过程中伴随着细胞外基质的硬化和YAP激活，提示YAP可能也参与OA病理过程。因此我们假说：OA软骨基质的病理力学影响软骨细胞表型并能作为治疗药物开发靶点。我们前期研究发现OA病理力学环境下YAP活性升高，进一步支持这一假说。因此，本研究将：①检测正常和OA组织ECM硬度和YAP活性改变；②在体外模拟构建不同硬度的基质培养软骨细胞，筛选最佳基质硬度，分析YAP在基质软硬度调控软骨细胞表型中的作用及机理；③用敲基因小鼠模型验证YAP在维持软骨稳态中的作用；④关节腔内缓释YAP小分子抑制剂分析其对阻缓骨关节炎病理进程的效应及作用机理。本研究发现将阐明关节软骨基质病理性的力学改变及YAP在其中的作用及机制，为骨关节炎的治疗提供新知识和新手段。

骨关节炎（OA）目前治疗药物多是处理炎症或疼痛等对症治疗，而OA的启动除炎症等因素，软骨基质力学有病理性改变。以往研究表明力学微环境影响细胞命运，肿瘤等疾病过程中伴随着细胞外基质的硬化和YAP激活，提示YAP可能也参与OA病理过程。因此我们假说：OA软骨基质的病理力学影响软骨细胞表型并能作为治疗药物开发靶点。我们前期研究发现OA病理力学环境下YAP活性升高，进一步支持这一假说。因此，本研究将：①检测正常和OA组织ECM硬度和YAP活性改变；②在体外模拟构建不同硬度的基质培养软骨细胞，筛选最佳基质硬度，分析YAP在基质软硬度调控软骨细胞表型中的作用及机理；③用敲基因小鼠模型验证YAP在维持软骨稳态中的作用；④关节腔内缓释YAP小分子抑制剂分析其对阻缓骨关节炎病理进程的效应及作用机理。通过本项目的研究，我们发现在人骨关节炎患者样本中有病理性ECM硬度增高，同时发现该病理性ECM硬度改变与YAP在细胞内活性增高关联。此外，我们利用PDMS材料体外构建不同软硬度的细胞培养基质可调控软骨细胞的表型，生理硬度范围的细胞培养基质有利于软骨细胞表型维持，该表型的改变同样与YAP在细胞内的活性相关。在条件性敲除YAP的小鼠模型上进行OA造模，骨性关节炎的进展得到延缓，提示我们YAP在关节软骨基质病理性的力学改变及其与OA进展的关系。体外运用YAP活性抑制剂维替泊芬可以维持软骨细胞表型，而关节腔注射YAP活性抑制剂维替泊芬可延缓骨性关节炎的进展。本研究发现阐明了关节软骨基质病理性的力学改变及YAP在其中的作用及机制，为骨关节炎的治疗提供了新知识和新手段。