Quantification of mRNA Using Real-Time PCR and Western Blot Analysis of MAPK Events in Chondrocyte/Agarose Constructs
Quantification of mRNA Using Real-Time PCR and Western Blot Analysis of MAPK Events in Chondrocyte/Agarose Constructs
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DOI：
10.1007/978-1-60761-984-0_6
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发表时间：
2011-01-01
期刊：
影响因子：
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通讯作者：
Chowdhury, Tina T.
中科院分区：
文献类型：
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作者：
Lee, David A.;Brand, June;Chowdhury, Tina T.
软骨细胞机械生物学的体外模型已被用来比较细胞内信号通路改变正常和骨关节炎影响的软骨。然而，模型系统和装载配置类型的差异导致了复杂的途径。本章是我们小组以前研究的后续，利用3D琼脂糖作为生理模型来研究机械转导通路。实验方法分别通过Western印迹分析和实时PCR在蛋白质和基因表达水平上评估靶点。本章提供了一个定量的基因表达的方法来探索细胞内的途径激活的机械负荷和炎症介质和检查上游磷酸化事件。最终，用于分析机械敏感途径的方法的发展将为确定用于治疗骨关节炎的适当药理学和物理治疗剂提供重要信息。
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 arc 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.