Interactions between cell adhesion and siganaling molecules in mechanical stress response of chondrocytes

生物力学力是决定骨骼组织形态和分化的主要表观遗传因素，并可能通过细胞粘附到细胞外基质（ECM）来传导。为了验证在早期软骨形成过程中逐步张力被转导成分子信号的假设，我们开发了一个研究软骨细胞增殖和分化的培养系统。将大鼠胚胎第12天的肢体芽进行微解剖并分离成细胞，然后在硅胶膜上进行微批量培养并维持6天。从第3天开始，对这些培养物逐步施加张力。分析软骨特异性标记物和非软骨性标记物的表达模式和水平的时间过程，并与非拉伸对照培养进行比较。在张力条件下，阿利新蓝染色显示软骨形成的速度和程度明显下降，共聚焦激光扫描显微镜显示细胞明显拉伸。从数量上看，在拉力加载12小时后，II型胶原和聚集蛋白的表达分别被显著抑制了20 ~ 30%和10 ~ 20%，这种差异在培养3天后保持不变。相比之下，I型胶原蛋白和纤维连接蛋白的表达在整个实验期间保持相对稳定。当GRGDSPK （Gly-Arg-Gly-Asp-Ser-Pro-Lys）肽抑制细胞ecm附着时，软骨标志物表达的下调被完全恢复。我们得出结论，逐步张力通过胚胎肢芽间质中的整合素抑制软骨形成，并提出生物力学刺激的信号转导可能通过细胞- ecm粘附介导。

Biomechanical forces are major epigenetic factors that determine the form and differentiation of skeletal tissues, and may be transduced by cell adhesion to the extracellular matrix (ECM). To test the hypothesis that stepwise tension forces are transduced into molecular signals during early chondrogenesis, we developed a culture system studying the proliferation and differentiation of chondrocytes. Rat embryonic day-12 limb buds were microdissected and dissociated into cells which were then micro-mass cultured on a silicone membrane and maintained for up to 6 days. Stepwise tension was applied to these cultures from day 3. The time course of the expression pattern and level of cartilage-specific markers and non-chondrogenic markers were analyzed and compared with those in non-stretched control cultures. Under tension conditions, Alcian blue staining showed an apparent decrease in the rate and extent of chondrogenesis, and confocal laser scanning microscopy demonstrated apparent stretching of the cells. Quantitatively, type II collagen and aggrecan expression were significantly inhibited by 20 to 30% and 10 to 20%, respectively, after 12 hrs of tension force loading, and this difference was maintained through 3 days of culture. In contrast, the expression of type I collagen and fibronectin remained relatively constant throughout the experimental period. This down-regulation in the expression of chondrogenic markers was completely rescued when cell-ECM attachment was inhibited by GRGDSPK (Gly-Arg-Gly-Asp-Ser-Pro-Lys) peptide. We conclude that stepwise tension inhibits chondrogenesis through integrins in embryonic limb bud mesenchyme, and propose that signal transduction from biomechanical stimuli may be mediated by cell-ECM adhesion.