YAP在应力调控生长板软骨细胞肥大分化中的作用及机制

Mechanical loading has been proved to be extensively important to the formation and development of growth plate. However, the regulatory mechanism of mechanical loading and related mechanotransduction are still not completely understood. It is found, in previous study, YAP (Yes-associated protein) was involved in the mechanotransduction of growth plate chondrocytes and we hypothesized that YAP may play an important role in the physiological development and pathological disorders of growth plate. In our studies, firstly, the growth plate chondrocytes will be obtained and culture in vitro. Then we will investigate the activation of YAP in growth plate chondrocytes which will be subjected to mechanical loading. Simutaneously, the expression of FAK（Focal adhesion kinase）, RhoA，F-actin and LINC (linker of nucleoskeleton and cytoskeleton) complex will be observed and detected. And the relationship between these possible signal transduction factors and YAP will be analyzed by using agonist or antagonist. Finally, the role of YAP in hypertrophic differentiation of growth plate chondrocytes in response to mechanical loading will be explored. In a word, the more we understand the mechanotransduction of the growth plate, the better we develop a new strategy for interventions of pathological growth plate in early stage.

应力对生长板的形成和发育至关重要，而力学对生长板软骨细胞的调控机制以及相应力学传导过程尚不明确。申请者在前期研究中发现YAP (Yes-associated protein)参与生长板软骨细胞力学信号传导过程，推测YAP在应力调控生长板正常发育和异常病变中发挥重要作用。本项目首先体外分离培养生长板软骨细胞；然后研究应力对生长板软骨细胞内YAP活性的调控作用；同时观察和检测FAK（Focal adhesion kinase）、RhoA、F-actin和LINC(linker of nucleoskeleton and cytoskeleton) complex的表达；并采用激动剂或拮抗剂，研究这些可能的信号传导因子和YAP之间的关系；最后我们研究YAP在力学调控生长板软骨细胞肥大分化中的作用。对生长板力学信号转导过程的认识越深，越能为早期干预生长板异常病变提供新方法。

应力对生长板的形成和发育至关重要，而力学对生长板软骨细胞的调控机制以及相应力学传导过程尚不明确。YAP（yes-associated protein）和LINC complex是重要的力学信号转导因子。本项目首先体外分离培养生长板软骨细胞，并将其与骨髓间充干细胞比较，观察两者成软骨分化能力;然后应力加载予以体外力学干预，观察和检测干预前后细胞骨架以及生长板软骨细胞肥大分化相关基因COL X和Ihh的表达；对可能的信号传导通路，予以拮抗剂或RNAi技术；最后采用新的评分量表来明确恶病质分期。.通过上述研究内容的开展，得出以下结果：1.体外分离培养的生长板软骨细胞增殖能力强于骨髓间充质干细胞，单独培养的生长板软骨细胞组和共培养组成软骨分化能力要强于单独培养的骨髓间充质干细胞组。2.短时间的应力能激活生长板软骨细胞内FAK、RhoA和YAP蛋白，并促进细胞骨架F-actin聚集，以及下调细胞肥大分化相关基因COL X和Ihh的表达。LINC complex抑制剂siSUN可以抑制应力对FAK、RhoA和YAP蛋白的活化、细胞骨架F-actin的聚集，以及COL X和Ihh的表达减少。3.长时间的应力能激活mTORC2、AKT和PKCα的磷酸化，以及上调COL X和Ihh的表达，PKCα抑制剂GO-6976或siRictor能抑制应力对COL X和Ihh的上调作用。4.新型分期评分量表充分验证了恶病质严重程度与肌肉减少症之间的关系。.上述研究表明三维共培养生长板软骨细胞和骨髓间充质干细胞可作为生长板修复和再生的理想种子细胞。YAP、LINC complex和mTORC2信号通路参与了力学信号传导过程，同时结果显示新型恶病质评分量表将有助于我们明确YAP和肌肉减少症的关系。