Wnt/DKK1负反馈对骨关节炎骨-软骨界面crosstalk的调控作用及其分子机制

Osteoarthritis (OA) is the most common human joint disease and is lack of efficient therapy. Our previous studies have suggested that an abnormal subchondral bone microstructure be intimately involved in the genesis of OA. Wnt/DKK1 negative feedback plays a vital role in the metabolism of articular cartilage and subchondral bone. Wnt/DKK1 negative feedback may regulate the bone-cartilage interface crosstalk between subchondral bone and articular cartilage in OA patients，but the effect and molecular mechanism are not clear. Therefore, in order to elucidate the effect and molecular mechanism of Wnt/DKK1 negative feedback regulating the bone-cartilage interface crosstalk between subchondral bone and articular cartilage, the direct co-culture model of subchondral bone osteoblasts (SBO) and articular cartilage chondrocytes (ACC) derived from OA patients and rat OA model will be used. We will investigate if the SBO-ACC crosstalk between OA SBO and OA ACC alters the phenotypes of SBO and ACC in vitro, and also test possible involvement of Wnt/DKK1 negative feedback signaling pathway by DKK1 gene transfer and DKK1 knockdown with small interfering RNA in SBO or ACC during this crosstalk process. In addition, we will investigate the in vivo effects of Wnt/DKK1 negative feedback regulating the bone-cartilage interface crosstalk between subchondral bone and articular cartilage on an experiment rat model of OA induced by anterior cruciate ligament transection (ACLT). Therefore, this research will provide direct insight into the effect and molecular mechanism underlying the cycle between subchondral bone and articular cartilage in the development of OA, and the altered SBO-ACC crosstalk between SBO and ACC may be mediated by Wnt/DKK1 negative feedback. This study might elucidate the effect and molecular mechanism of Wnt/DKK1 negative feedback regulating the bone-cartilage interface crosstalk between subchondral bone and articular cartilage. The Wnt/DKK1 negative feedback might be a new potential therapeutic target for efficient treatment of OA，and this research will be important advanced research for efficient therapeutic strategies of OA.

骨关节炎（OA）是一种严重危害中老年人健康、目前尚无有效治疗手段的慢性退变性骨关节病变。我们的前期研究发现软骨下骨的微观结构异常与OA的发生和进展密切相关。Wnt/DKK1负反馈是参与调控骨与软骨代谢的重要通路， Wnt/DKK1负反馈可调控OA骨-软骨界面串扰(crosstalk)，但其机制尚未阐明。本项目拟采用SBO与ACC直接共培养和大鼠OA动物模型；体外探讨上调和下调SBO或ACC中DKK1表达对OA患者SBO-ACC crosstalk的影响以及 Wnt/DKK1负反馈调控SBO-ACC crosstalk的分子机制；体内探讨Wnt/DKK1负反馈对OA骨-软骨界面crosstalk的调控机制。研究成果将可能阐明Wnt/DKK1负反馈调控OA骨-软骨界面crosstalk的分子机制。Wnt/DKK1负反馈可能是有效治疗OA的潜在治疗靶点，本研究成果将为OA的治疗提供重要的进展。

随着人口老龄化的日益加剧，骨性关节炎（OA）成为影响人民健康的主要慢性退行性疾病，而且目前尚无有效治疗手段。已有研究表明Wnt/DKK1系统是参与调控骨与软骨代谢的重要通路，与OA软骨下骨的骨重构及OA的发生进展密切相关，但Wnt/DKK1系统调控OA软骨下骨成骨细胞及关节软骨细胞的相互作用的机制尚未阐明。该研究成功地从人及SD大鼠中成功提取出成骨细胞及关节软骨细胞，设计构建纯化的过表达DKK1的重组慢病毒。鉴于从OA患者关节组织提取出的软骨细胞状态较差，生长缓慢，我们选用具有类似细胞功能的人源SW1353细胞系代替，构建过表达DKK1的稳转株。利用Transwell培养板构建共培养体系，我们发现成骨细胞与炎性软骨细胞共培养后，成骨细胞的成骨活动增强，而这种作用是激活了成骨细胞内经典Wnt信号通路引起，与过表达DKK1的软骨细胞共培养后，能够有效抑制成骨活动。另外，在体外培养出大鼠的关节软骨细胞及成骨细胞，分别使用大鼠重组白细胞介素-1β干预软骨细胞，使用siRNA干扰技术敲低大鼠成骨细胞中的经典Wnt信号通路核心分子β-catenin，我们发现抑制经典Wnt信号通路能够有效缓解白介素-1β引起的软骨细胞外基质降解，并在成骨细胞的成骨活动中有不可或缺的作用。最后，我们切除SD大鼠膝关节前十字韧带构建大鼠OA模型，利用微型计算机断层扫描、关节软骨染色及免疫组化等技术，我们发现大鼠膝关节骨性关节炎在发生进展过程中，软骨下骨出现BV/TV和骨小梁数目减少，小梁分离增加等骨重塑现象。总之，该研究体外研究了经典Wnt信号通路在软骨细胞、成骨细胞中的作用，并成功构建软骨细胞与成骨细胞的共培养体系，结合重组慢病毒技术，探讨了Wnt/DKK1系统参与调控骨与软骨代谢的重要机制；在体内大鼠OA模型中，并初步探索了在体内缓解骨性关节炎软骨退变对软骨下骨骨重塑的影响，为OA治疗策略提供重要进展。