从Wnt通路靶向调节OPN探讨骨关节炎发病的分子机制

The current concept holds that osteoarthritis (OA) involves not just the articular cartilage but the entire joint organ, including the synovium and subchondral bone. Wnt/β-catenin signaling is associated with chondrocyte function and behavior. Our previous study revealed WIF-1 and β-catenin correlated with the degree of cartilage degeneration. Osteopontin (OPN) is a phosphorylated acidic glycoprotein. We also had showed OA patients had high OPN expression in synovium and articular cartilage. Moreover, the previous study indicated expression of osteopontin, a target gene of de-regulated Wnt signaling, predicted survival in colon cancer. However, the molecular mechanisms that regulate the expression of OPN are incompletely understood by Wnt signaling in OA and it is not clear if they are very good target for OA therapy. We hypothesized that OPN expression can be regulated by the Wnt pathway in OA and small interfering RNA (siRNA)-mediated inhibition of post-transcriptional events can delay or prevent the progression of OA. To prove this hypothesis, we examine the expression of Wnt pathway components and OPN in joint tissues, chondrocyte and synovial cell from OA patients firstly. Secondly, we elucidate the effect and mechanism of Wnt pathway regulating OPN and inducing osteoarthritis-like changes of joint. Finally, we identify the effects of siRNA towards its target site on OA. Consequently, the present study elucidates the pathologic progression and mechanisms of OA from a new perspective and provides new target gene for new treatment against OA.

骨关节炎(OA)是一种累及软骨、滑膜和软骨下骨等结构的疾病。Wnt/β-catenin通路与软骨细胞功能行为等密切相关，我们曾证实Wnt抑制因子1、β-catenin与软骨退变程度相关。OPN是一种糖蛋白，我们也证实其在OA患者软骨和滑膜中高表达。先前研究提示，Wnt通路调控OPN预测结肠癌的生存率。但Wnt通路和OPN在OA中的具体调控机制及其是否是OA有效治疗靶点尚未明确。我们推测OA中Wnt通路对OPN有一定调控作用，且在其作用途径中给予siRNA干扰可能延缓甚至阻止OA的进展。为证实这一假说，我们采用EMSA、siRNA等分子生物学技术，观察关节内组织和细胞中Wnt通路成员和OPN的表达，探讨Wnt通路靶向调控OPN介导关节OA改变的作用和机制，明确靶基因siRNA对OA的效应。本课题将从新的视觉阐明OA的发病和发展机制，为探寻OA新的治疗方法提供新的靶分子。

骨关节炎(OA)是一种常见退行性关节疾病。我们已证实Wnt通路和骨桥蛋白（OPN）与OA发病相关。先前研究提示，Wnt通路调控OPN预测结肠癌的生存率。故推测OA中Wnt通路对OPN有一定调控作用，且在其作用途径中给予siRNA干扰可能延缓甚至阻止OA的进展。为了证实以上推测：首先，运用免疫组化法观察Wnt通路和OPN在OA软骨中的表达及其与病情严重性的相关性。结果显示：DKK-1、Wnt3A、TCF-4、WISP-1、β-catenin、αvβ3、PI3K、OPN、β-半乳糖苷酶在OA软骨中异常表达。WISP-1、β-catenin、αvβ3、OPN、β-半乳糖苷酶与OA病情严重性呈正相关。第二，WB和qRT-PCR方法检测OA患者软骨细胞中Wnt通路和OPN表达水平，结果与免疫组化一致。第三，通过凝胶迁移试验和荧光酶素活性分析，发现TCF-4与β-catenin存在结合，并调节OPN的表达。第四，运用过表达质粒或siRNA转染人软骨细胞，发现升高和降低TCF-4表达分别引起OPN表达的升高和降低；发现升高β-catenin表达引起OPN表达升高，但降低β-catenin表达不改变OPN表达。此外，通过上调和下调软骨细胞WISP-1表达观察WISP-1的作用，并通过αvβ3 抗体和PI3K/Akt抑制剂观察WISP-1的作用机制，发现WISP-1与αvβ3受体结合，激活PI3K/Akt通路，上调OPN表达并抑制软骨细胞凋亡和衰老。第五，OA动物模型关节内注射ICG-001降低β-catenin表达后，引起OPN的表达下调、软骨细胞凋亡增加。第六，OA动物模型关节内注射LiCI升高WISP1表达后，引起OPN表达的升高，软骨细胞衰老和凋亡的降低，进一步缓解OA进展。而WISP1siRNA干预见到相反的结果。本项目首次发现异常表达的Wnt通路成员（如WISP-1）与OA严重程度相关，可以用于监测OA病情进展。升高的β-catenin与TCF-4结合，形成β-catenin/TCF-4 复合物，激活OPN表达，影响软骨细胞凋亡，参与OA发生发展。并且，WISP-1结合αvβ3后，激活PI3K/Akt通路，促进OPN表达，抑制软骨细胞凋亡和衰老，缓解OA进展。本课题从新的视觉阐明了OA的发病和发展机制，为探寻OA治疗提供了新的靶分子。