不同条件运动负荷下关节软骨细胞表型的变化及相关机制

Osteoarthritis (OA) is a degenerative joint disease characterized by the change of chondrocyte phenotype and the matrix being mineralized. There is a large number of study regarding the relation between exercise and OA, but lack of the study in molecular level. TRPV5、TRPV6, the new type of calcium ion channel, is important to maintain calcium balance in vivo, which may play a role in cartilage matrix mineralization. The normal and OA SD rat are enforced to exercise under different conditions. 2-dimensional gel electrophoresis (2-DE) is used to investigate the differentially expressed proteins belong to chondrocyte phenotype. Then immunohistochemistry technique and confocal laser electronic microscope technique are used to detect the change of specified chondrocyte phenotype, including collagen type II and Ⅹ, aggrecan, Sox9, MMP-13, ALP, TRPV5 and TRPV6. Finally, the real time RT-PCR is used to measure the change of specified chondrocyte phenotype above in gene level. Simultaneously, the chondrocyte separated from normal SD rat and the animal model rat is cultured. Then the change of the chondrocyte phenotype, including collagen type II and Ⅹ, aggrecan, Sox9, MMP-13, ALP, before/after gene knockout of TRPV5、TRPV6 is investigated using real time RT-PCR technique. Thus the relation between exercise and OA and the relevant mechanism can be studied deeply, and the role of TRPV5、TRPV6 in OA can be determined explicitly, which can not only deepen the knowledge of the mechanism of OA, but guide the sports exercises and rehabilitation training scientificlyv to relieve the OA patient's condition and reduce the morbidity of OA.

骨关节炎(OA)是导致中老年人慢性残障最常见原因，病理学改变为软骨细胞正常表型丢失、基质矿化。运动与OA关系日益受到重视，但缺乏分子水平深入研究。新型钙离子通道TRPV5、TRPV6对维持体内钙平衡至关重要，对软骨基质代谢的影响尚属未知。本研究建立SD正常及OA大鼠不同条件运动负荷动物模型，采用双向电泳技术筛选软骨细胞差异表达的蛋白表型，免疫组化技术、激光共聚焦电镜技术观察特定细胞表型表达变化，而后实时RT-PCR技术从基因水平对软骨细胞表型蛋白质的变化进行分析。同时，分离培养正常和运动负荷动物模型大鼠膝关节软骨细胞，观察TRPV5、TRPV6基因敲除前后软骨细胞表型表达变化。深入探讨运动负荷与OA关系及相关机理，明确TRPV5、TRPV6在OA发病中的作用，加深对OA发病机制的认识，不但为OA对因治疗学提供重要参考，也可指导科学的进行运动和康复训练，缓解OA患者病情，降低OA发病率。

骨性关节炎(osteoarthritis，OA)是导致中老年人慢性残障的最常见原因,预测到2030年将会成为世界老年致残的第一大疾病,严重降低患者的生活质量。在骨关节发生时，软骨细胞的凋亡被激活，自噬被抑制。我们发现在骨关节炎发生时，TRPV5表达量增加， TRPV5过表达能够介导钙离子内流增加，内流的钙离子与calmodulin蛋白结合，启动下游级联反应。进而可以诱导软骨细胞凋亡，抑制软骨细胞自噬。我们还对其内在的具体机制作了探讨。为骨关节炎的治疗提供新的视点。不同运动负荷对软骨细胞表型的影响也是本课题中深刻探讨的问题。我们揭示了不同运动负荷下软骨细胞Ⅱ型胶原会出现不同的变化，进而反映软骨细胞损伤情况，这一研究为指导合理运动提供依据。运动对骨关节炎的作用一直是争论的焦点，此外我们通过构建骨关节炎运动负荷模型具有新的发现：不同运动负荷对LXA4代谢的影响，揭示了LXA4代谢的变化在运动治疗骨性关节炎中发挥的作用，在保证其升高的情况下尽可能减少过度压力的刺激，为制定合理运动提供更有效检测指标。