Whole transcriptome analysis for ossification of vertebral ligament using RNA interference and microbeads array

人骨化脊椎韧带细胞信号和转录因子表达的组织学、免疫组织化学和实时RT-PCR分析。我们分析了在周期性拉伸应变下培养的骨化韧带细胞中已知参与骨化过程的信号因子的mRNA表达水平。FLEXHERS(R)FX-3000产生循环拉伸应变。用免疫组织化学方法检测这些因子在脱钙石蜡切片中的定位。对照样本取自患者的非骨化脊椎韧带。在静息状态下(无张应力)，培养的骨化韧带细胞β-连接素、Runx2、Sox9和骨桥蛋白的表达水平显著高于对照组。对骨化细胞施加循环拉伸应变后24小时，β-连环蛋白、Runx2、Sox9和骨桥蛋白的表达水平显著升高。钙化前沿周围肥大的软骨细胞表达Runx2和骨桥蛋白。β-连环蛋白和Sox9在纤维软骨区的早熟软骨细胞中呈强阳性表达。我们的结果表明，循环拉伸应变作用于骨化的脊椎韧带细胞，通过β-连环蛋白信号通路介导的过程激活其骨化。

Histological, immunohistochemical, and real-time RT-PCR analyses of the expression of cell signaling and transcriptional factors in human ossified vertebral ligament. We analyzed the mRNA expression levels of signaling factors known to be involved in the ossification process in cultured ossified ligament cells subjected to cyclic tensile strain. Cyclic tensile strain was produced by Flexerce(R) FX-3000. The localization of these factors was examined in decalcified paraffin sections by immunohistochemistry. Controlled samples were harvested from non-ossified vertebral ligament of patients. Under resting conditions(no tensile strain), the mRNA levels ofβ-catenin, Runx2, Sox9 and Osteopontin in cultured ossified ligament cells were significantly higher than in the control cells. Application of cyclic tensile strain to ossified cells resulted in significant increases in mRNA expression levels ofβ-catenin, Runx2, Sox9, and Osteopontin at 24 hours. Hypertrophic chondrocytes present around the calcification front were immunopositive for Runx2 and Osteopontin. Immunoreactivity ofβ-catenin and Sox9 was strongly present in premature chondrocytes in the fibrocartilage area. Our results indicated that cyclic tensile strain applied to ossified vertebral ligament cells activated their ossification through a process mediated by theβ-catenin signaling pathway.