EphB4/ephrinB2在关节软骨细胞终末分化及骨性关节炎发病中的作用

Chondrocyte terminal differentiation involved in the occurrence and development of osteoarthritis. However, the mechanism of articular chondrocyte terminal differentiation is still unclear currently. Based on previous findings, tyrosine kinase receptor EphB4 and ligand ephrinB2 interaction involve in chondrocyte terminal differentiation and in the endochondral ossification of growth plate and fracture healing. We postulate that EphB4/ephrinB2 involve in the regulation of osteoarthritic chondrocyte terminal differentiation. Firstly, the present study will observe the expression of EphB4 and ephrinB2, the markers of chondrocyte terminal differentiation collagen X, MMP-13 and ALP in the rat medial meniscal tear model of osteoarthritis. Secondly, to evaluate the role of exogenous ephrinB2 and TNYL-RAW, a specific peptide antagonist of EphB4/ephrinB2 interaction on the three-dimensional cultured chondrocyte of rat knee joint based on the expression level of collagen X, MMP-13, ALP, and the changes of chondrocyte proliferation, hypertrophy, apoptosis, and analyze the potential signaling pathways involved; Finally, to explore the effect of Ad/EphB4 and Ad/siRNA/EphB4 transfection on articular chondrocyte differentiation in vitro and the effect on cartilage degeneration in rat osteoarthritis model. The present study aims to clarify the role of EphB4/ephrinB2 in the regulation of articular chondrocyte terminal differentiation and in the pathogenesis of osteoarthritis, expecting to find new ways in the prevention and treatment of osteoarthritis.

关节软骨细胞终末分化参与骨性关节炎（OA）的发生和发展，但关节软骨细胞终末分化的机制目前所知甚少。最近有研究表明，酪氨酸蛋白激酶受体EphB4与其配体ephrinB2参与生长板与骨折愈合的软骨细胞终末分化和软骨内骨化。我们的前期研究发现EphB4在OA关节软骨中表达较正常明显升高。因而，本课题拟进一步观察EphB4/ephrinB2在关节软骨细胞终末分化及OA发病中的作用。课题首先检测大鼠膝关节退变关节软骨内EphB4/ephrinB2、软骨细胞终末分化标记物X型胶原/MMP-13/ALP的表达变化；然后观察外源性ephrinB2与EphB4/ephrinB2阻断剂TNYL-RAW对培养的大鼠膝关节软骨细胞增殖、肥大、凋亡以及终末分化标记物表达的影响，并分析可能参与的信号传导途径；最后通过体内、外实验观察腺病毒EphB4和EphB4 siRNA对关节软骨细胞终末分化和OA病理过程的影响。

本项目通过体内实验观察到酪氨酸蛋白激酶受体EphB4与其配体ephrinB2参与参与了半月板撕裂所致大鼠膝关节骨性关节炎的病理过程，外源性ephrinB2 可以增加软骨细胞软骨基质II型胶原的表达，降低软骨细胞终末分化标志物MMP-13和COL-X的表达，软骨细胞凋亡减少，而EphB4/ephrinB2 特异性阻断剂TNYL-RAW肽降低软骨细胞软骨基质II型胶原的表达，促进软骨细胞终末分化标志物MMP-13和COL-X的表达，软骨细胞凋亡增加，MAPKs和PI3K-Akt信号通路均有参与这一过程。采用大鼠骨性关节炎模型体内实验观察到腺病毒Ad/siRNA/EphB4关节腔注射后可以减缓关节软骨退变。该初步研究结果表明EphB4/ephrinB2参与关节软骨细胞终末分化过程，在骨关节炎的发生发展中发挥作用，进一步深化对骨性关节炎病理机制的理解。.另外本项目通过大鼠骨关节炎模型观察到，骨关节炎软骨下骨转换活跃，骨的代谢、结构、数/质量和生物力学性能均发生了显著的动态性改变，病情早期软骨下小梁骨的BV/TV、Tb.Th降低，Tb.Sp增加，随后时小梁骨的BV/TV、Tb.Th增加，Tb.Sp下降，观察期内Tb.N、Cnn.D持续减少，12周时BP.Th增加；骨板和小梁骨的TMD、PO43-/proline、Er和H均随病程进展持续下降；相对于软骨下骨板，骨小梁变化趋势更为明显。早期给予唑来膦酸治疗可以抑制软骨下骨改建，延缓关节退变，减轻关节负重性疼痛，降低疼痛标志物的表达。类似的，给予雷尼酸锶治疗能够减少软骨损伤，降低软骨细胞凋亡；改善软骨下骨的显微结构，增加PO43-/proline，提高Er和H。这些研究结果进一步明确了软骨下骨在OA软骨退变与关节疼痛防治中的作用；为骨保护类药物在OA临床中的应用提供科学依据。