Lee David A, Brand June, Salter Donald, Akanji Oto-Ola, Chowdhury Tina T
School of Engineering and Materials Science, Queen Mary University of London, London, UK.
Methods Mol Biol. 2011;695:77-97. doi: 10.1007/978-1-60761-984-0_6.
In vitro models of chondrocyte mechanobiology have been used to compare the intracellular signalling pathways altered in normal and osteoarthritis-affected cartilage. However, differences in the model system and type of loading configuration have led to complicated pathways. This chapter is a follow-on of previous studies from our group utilising 3D agarose as a physiological model to study mechanotransduction pathways. Experimental methods are described to assess targets at the protein and gene expression level by Western blot analysis and real-time PCR, respectively. This chapter provides a quantitative gene expression approach to explore the intracellular pathways activated by both mechanical loading and inflammatory mediators and examine upstream phosphorylation events. Ultimately, development of methods used to analyse mechano-sensitive pathways will provide important information for the identification of appropriate pharmacological and physiotherapeutic agents for the treatment of osteoarthritis.
软骨细胞力学生物学的体外模型已被用于比较正常软骨和骨关节炎影响的软骨中改变的细胞内信号通路。然而，模型系统和加载配置类型的差异导致了复杂的信号通路。本章是我们小组先前研究的延续，利用三维琼脂糖作为生理模型来研究机械转导通路。分别描述了通过蛋白质印迹分析和实时聚合酶链反应在蛋白质和基因表达水平评估靶点的实验方法。本章提供了一种定量基因表达方法，以探索由机械加载和炎症介质激活的细胞内信号通路，并检测上游磷酸化事件。最终，用于分析机械敏感信号通路的方法的开发将为识别用于治疗骨关节炎的合适药物和物理治疗药物提供重要信息。
