2013 年 11 巻 1 号 p. 29-41
Previously we reported a successful three-dimensional cartilage regeneration using an RWV (rotation wall vessel) bioreactor from human mesenchymal stem cells (hMSCs)(Sakai et al. J Orthop Res. 27, 2009), which is useful for better understanding the initiation mechanism of chondrogenic differentiation of hMSCs and discovering its associated proteins. Entire proteome of hMSCs and spherical cartilaginous constructs by RWV culture was comparatively analyzed using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization/ time-of-flight/mass spectrometry (MALDI-TOF-MS/MS) to determine candidate proteins associated with early chondrogenesis. Among them, there found a greatly up-regulation of COL6A1 and COL6A2 proteins on day 10, which encode matrix proteins with great changes at early stage of chondrogenesis in parallel with the increase of SOX9. A specific antibody against to the extracellular protein COL6 effectively postponed the progress of early chondrogenesis, followed by the successive reduction of extracellular COL6 in spite of a temporary increase in genes expression and up-regulation of SOX9 phosphorylation on the early stage of antibody neutralization. It suggested that the extracellular matrix protein COL6 might be a potential key molecule to involve in early events of chondrogenesis via regulating or influencing the expression of SOX9.