骨桥蛋白激活NF-κB信号通路调控OA滑膜细胞MMP13表达的实验研究

发布时间：2019-02-16 08:19

【摘要】：目的:骨性关节炎(Osteoarthritis,OA)是一种常见的慢性关节病。其发病率随着年龄增长逐渐增加。其病理特征为原发或继发性的关节软骨病变,具体表现为关节软骨胶原蛋白降解、蛋白多糖降解和关节软骨结构破坏,同时也会导致一定程度的滑膜炎症。WHO数据显示,10%男性及18女性%以及65%年龄超过60岁的人都患有不同程度的骨关节炎。虽然OA的病因是比较复杂的(遗传,体质,生物力学因素),但我们对OA的发病机制的认识正不断加深。骨桥蛋白(Osteopontin,OPN)是一种广泛分布于人体组织中,具有多种功能的磷酸化蛋白。OPN可由多种细胞分泌,包括巨噬细胞、淋巴细胞、上皮细胞、血管平滑肌细胞、滑膜细胞以及软骨细胞。在骨组织中,OPN可调节细胞基质和细胞间的活动,参与骨折修复中软骨向骨的转化以及破骨细胞对骨基质的附着。骨桥蛋白(Osteopontin,OPN)与骨关节炎的严重程度及疾病的发生进展密切相关,但OPN在OA发病机制中的作用尚未明确。本研究目的旨在探讨骨桥蛋白在膝骨关节炎(Osteoarthritis,OA)中的调控作用以及涉及的信号转导通路。方法:1.选取15名OA患者其中男性7例,女性8例,平均年龄61.2±8.5岁。选取同期正常对照组15例,为不伴有骨关节炎的半月板损伤或韧带损伤。对照组男性9例,女性6例,平均年龄42.4±7.8岁。Ⅱ型胶原酶一步消化法处理OA滑膜组织,分离培养OA软骨细胞并传代,取第一代OA软骨细胞继续培养。2.根据文献报道siRNA序列,合成OPN-siRNA,与OPN组作为实验组,无意义序列Con-siRNA为阴性对照组,将两组序列进行脂质体转染,采用Western Blotting检测NF-κB(nuclear factor-KappaB)p65、p-NF-κB p65的蛋白水平,将得到的Western blot图片,用IPP软件处理,得出灰密度值,进行目的蛋白NF-κB/p-NF-κB的均一化处理和内参蛋白β-actin的均一化处理,将浓度组得到的NF-κB/p-NF-κB p65的均一化结果除以β-actin的结果,得到各组的相对定量值。3.以转染Con-siRNA的OA滑膜细胞为对照组,转染OPN-siRNA、OPN、PDTC及PDTC联合OPN处理细胞为实验组。用Western blot检测MMP13表达情况。用上述方法得到各组相对定量值。结果:相比对照组,经OPN干预后,P65含量及P65磷酸化显著增加(P=0.0025,P=0.0019)。然而,OPN-siRNA处理后的P65含量及P65磷酸化相比对照组显著降低(P=0.0024,P=0.0176)。OPN可显著提高MMP13蛋白含量(P=0.0075),而OPN-siRNA可显著降低蛋白含量(P=0.0021),NF-κB抑制剂(PDTC)可明显拮抗OPN对MMP13蛋白的调控(P=0.0231)。结论:OPN可促进OA滑膜细胞NF-κB p65、p-NF-κB p65的蛋白表达,并通过激活NF-κB信号通路,促进MMP13蛋白表达。这可能是OA发病机理的重要组成部分。
[Abstract]:Objective: osteoarthritis (Osteoarthritis,OA) is a common chronic arthropathy. The incidence rate increases with age. The pathological features are primary or secondary articular cartilage lesions, including collagen degradation of articular cartilage, degradation of proteoglycan and destruction of articular cartilage structure, as well as synovitis to a certain extent. WHO data show that, Ten percent of men and 18 percent of women and 65 percent of people over 60 had varying degrees of osteoarthritis. Although the etiology of OA is complex (genetic, physical, biomechanical factors), our understanding of the pathogenesis of OA is deepening. Osteopontin (Osteopontin,OPN) is a phosphorylated protein widely distributed in human tissues. OPN can be secreted by a variety of cells, including macrophages, lymphocytes, epithelial cells, vascular smooth muscle cells. Synovial cells and chondrocytes. In bone tissue, OPN can regulate the activity of cell matrix and cells, participate in the transformation of cartilage to bone and the attachment of osteoclasts to bone matrix in fracture repair. Osteopontin (Osteopontin,OPN) is closely related to the severity and progression of osteoarthritis, but the role of OPN in the pathogenesis of OA is not clear. The aim of this study was to investigate the regulatory role of osteopontin in knee osteoarthritis (Osteoarthritis,OA) and the signal transduction pathways involved. Methods: 1. 15 patients with OA were selected, including 7 males and 8 females, with an average age of 61.2 卤8.5 years. 15 cases of normal control group were selected as meniscus injury or ligament injury without osteoarthritis. There were 9 males and 6 females in the control group, with an average age of 42.4 卤7.8.Type 鈪，

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