Quantification of mRNA using real-time PCR and Western blot analysis of MAPK events in chondrocyte/agarose constructs.
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.
School of Engineering and Materials Science, Queen Mary University of London, London, UK.
David A
DA
Lee
June
J
Brand
Donald
D
Salter
Oto-Ola
OO
Akanji
Tina T
TT
Chowdhury
D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002417:Cattle; D002469:Cell Separation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000201:enzymology*; D018076:DNA, Complementary / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D053583:Interleukin-1beta / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012685:Sepharose / Q000737:chemistry*; D013997:Time Factors
D018076:DNA, Complementary; D053583:Interleukin-1beta; D012333:RNA, Messenger; D012685:Sepharose; D052247:Nitric Oxide Synthase Type II; D020928:Mitogen-Activated Protein Kinases; D012194:RNA-Directed DNA Polymerase