周期性机械应力促进软骨细胞增殖的Cav-1/IGF-1R信号研究

Periodic mechanical stress could significantly promote chodrocyte proliferation. However, the mechanisms underlying the ability of chondrocytes to detect and respond to periodic mechanical stress have not been well delineated. The key to clarify the above problem is to find the mechanosensors on the cell membrane surface, and the latters translate mechanical inputs into biochemical signaling events within the chondrocyte lineage. Our preliminary studies and literature revealed that Cav-1 and IGF-1R, which locate on the cell membrane surface, is involved in the ability of cells to detect and respond to extracellular mechanical environment. However, the exact mechanisms by which Cav-1 and IGF-1R participate in the biological activity and function of cells are barely known. We check the activation and interaction of Cav-1 and IGF-1R under periodic mechanical stress in this study firstly. Then we adopt the selective inhibitors, shRNA and point mutation techniques to inhibit the activation of the above signaling proteins, and test chonrocyte proliferation, the activation and interaction of the above Cav-1 and IGF-1R, to determine the role of the signaling proteins, including the nature of any functional association between the signaling proteins. This study was designed to determine the mechanism of signal transduction and search the non-integrin mechanosensors by which periodic mechanical stress promotes chondrocyte proliferation. This study will lay the foundation for further investigations regarding chondrocye mechanobiology under periodic mechanical stress and how to improve the quality of tissue-engineered cartilage.

周期性机械应力能促进软骨细胞增殖，但其机制尚不清楚，阐明上述问题的关键在于寻找细胞膜表面机械感受器，后者将机械力学刺激转化为生物化学信号。本课题组前期研究和文献报道表明，Cav-1和IGF-1R位于细胞膜表面，与细胞感知和应答细胞外机械力学环境密切联系，但参与机制仍缺乏研究。本课题拟首先观察周期性机械应力下Cav-1和IGF-1R的活化水平和相互作用，然后通过应用特异性抑制剂、shRNA和点突变技术等手段下调相应信号蛋白的活性，进一步观察细胞增殖、上述信号蛋白的活化水平和相互作用，以确定Cav-1和IGF-1R作为非整合素机械感受器在周期性机械应力下如何发挥重要作用，以及彼此之间的相互关系。本研究旨在探讨周期性应力促进软骨细胞增殖的信号机制，寻找细胞膜表面非整合素机械感受器，为进一步深入探讨如何提高组织工程软骨的质量奠定基础。

骨性关节炎引起的关节软骨损伤和创伤性关节软骨缺损是骨科常见疾患，其临床治疗一直是个难题。组织工程软骨是治疗和修复关节软骨损伤最有希望的方法之一。周期性机械应力可以模拟关节软骨周围生理力学环境，显著促进软骨细胞增殖，提高种子细胞的功能，因而在构建符合生理需要的优质组织工程软骨过程中得到广泛应用。然而，对于周期性机械应力促进软骨细胞增殖、构建优质组织工程软骨的内在相关机制，尚缺乏深入的研究。.阐明上述问题的关键在于寻找细胞膜表面的机械感受器，后者将机械力学刺激转化为生物化学信号。本项目在前期原有工作的基础上，进一步探讨非整合素机械感受器Cav-1和IGF-1R在周期性机械应力促进软骨细胞增殖过程中的重要作用。本课题首先观察周期性机械应力下Cav-1和IGF-1R的活化水平，然后通过应用特异性抑制剂和shRNA技术等手段下调相应信号蛋白的活性，进一步观察细胞增殖、Cav-1和IGF-1R以及下游信号蛋白ERK1/2的活化水平，以确定Cav-1和IGF-1R作为非整合素机械感受器在周期性机械应力下如何发挥重要作用，以及彼此之间的相互关系。本研究结果证实，周期性机械应力首先诱导非整合素机械感受器Cav-1的磷酸化，从而激活IGF-1R，活化的IGF-1R最终介导软骨细胞中的ERK1/2有丝分裂信号级联反应。因此，Cav-1-IGF-1R-ERK1/2构成了周期性机械应力促进软骨细胞增殖的一种关键信号转导途径。.周期性机械刺激是影响组织工程软骨质量的主要决定因素。因此，阐明周期性机械应力下软骨细胞增殖的信号传导通路至关重要。我们的课题成果将为进一步研究周期性机械应力下软骨细胞生理力学和如何提高组织工程软骨的质量奠定实验理论基础和提供新的的视角。