脂肪因子-visfatin及脂联素在骨关节炎中的作用机制研究

【作者】 段圆慧；

【导师】 邱贵兴；

【作者基本信息】 北京协和医学院 ， 骨外科学， 2011， 博士

【摘要】 目的1.分析visfatin在骨关节炎(OA)患者和正常对照组的血浆和关节液中的分布,探讨visfatin在OA中的作用；2.着重了解脂联素对OA软骨细胞的作用。方法ELISA方法测定30例女性OA患者和12例正常对照组血浆及关节液中visfatin,以及关节液中的软骨基质降解标记物,并对OA严重程度放射学分级,进行相关分析；应用细胞免疫荧光、Real-time PCR、western blotting技术,分析OA软骨细胞表达脂联素受体与对照组之间的差异；体外培养OA软骨细胞进行药物干预,观察脂联素对软骨细胞NO合成的影响。结果OA患者关节液visfatin浓度明显高于对照组,两组血浆visfatin浓度无统计学意义；相关分析显示,OA关节液visfatin与II型胶原降解标记物CTX-II及aggrecan降解标记物AGG1,AGG2水平正相关；脂联素受体在OA关节软骨细胞中的表达状况,发现与对照组相比,脂联素受体表达水平下调,其中AdipoRl下调最显著；体外实验证实脂联素能够活化NF-κB转录活性,引起iNOS启动子区域增强iNOS转录水平,最终导致iNOS蛋白表达增强,软骨细胞从而合成大量的NO水平,同时检测培养上清中细胞因子水平,发现脂联素能够明显增加细胞培养基中IL-6、TNF-α、MMP-3、MMP-9和TIMP-2水平,但不能增加IL-1β、TIMP-1、MCP-1、PGE2和MMP一13水平。结论1.visfatin是新发现的影响OA关节软骨代谢的又一脂肪因子,参与软骨基质的降解；2.脂联素能够促进软骨细胞合成释放某些炎症因子和l软骨基质代谢相关分子,可能是一种前炎症因子,发挥炎症效应,在OA病理生理过程中具有重要作用。这些结果表明了脂肪因子在OA中具有重要的代谢角色。更多还原

【Abstract】 Objective 1. To analyze the distribution of visfatin between plasma and synovial fluid (SF) of patients with osteoarthritis (OA) and normal subjects, identify the role of visfatin in OA.2. Focus on the effects of adiponectin on the chondrocytes in OA.Methods Levels of visfatin were measured by a sandwich enzyme-linked immunosorbent assay in plasma and SF collected from 30 female OA patients and 12 normal subjects. Degradation markers of cartilage matrix in SF were also measured. The severity of OA was determined according to the Kellgren and Lawrence classification. Correlation analyses between visfatin and degradation markers or OA severity were performed. Immunofluorescence, Real-time PCR, western blotting techniques were evaluated for the difference of adiponectin receptors expression between OA chondrocytes and normal chondrocytes; OA chondrocytes were stimulated with some inhibitors; effects of adiponectin on niric oxide synthesis in OA chondrocytes were observed in vitro.Results Compared to controls, OA patients had higher SF visfatin concentration (8.95±2.5 vs.4.48±2.49 ng/ml, P< 0.001). Plasma visfatin levelswere higher inOApatients than those in control subjects, but not statistical significance (5.37±1.45 vs. 4.93±1.4 ng/ml, P= 0.466). SF visfatin positively correlated with degradation biomarker of collagenⅡ, CTX-Ⅱ(r= 0.497, P= 0.005), and degradation biomarker of aggrecan, AGG1 (r=0.451, P=0.012) and AGG2 (r= 0.434, P=0.017). We compared the expression of adiponectin receptors in primary chondrocytes from OA patients and normal subjects, but compared to normal chondrocytes, the three adiponectin receptors were down-regulated in osteoarthritic chondrocytes, especially AdipoRl. In vitro experiments confirmed that adiponectin can activate the transcriptional activity of NF-κB, enhance promoter region of iNOS transcription, lead to increased expression of iNOS protein, and OA chondrocytes synthesed a large number of NO. In addition, adiponectin is able to increase IL-6, TNF-α, MMP-3, MMP-9 and TIMP-2 by cultured OA chondrocytes in vitro, whereas it was unable to modulate IL-1β, MCP-1, PGE2, TIMP-1 and MMP-13 release.Conclusion 1. visfatin is a new discovery of another adipokines, regulating the degradation of cartilage matrix in OA.2. Adiponectin can release certain inflammatory cytokines and cartilage matrix metabolism molecules by OA chondrocytes, may be a proinflammatory factor, had an important role in the pathogenesis of OA. These results suggest that fat-derived adipokines have an important role in the metabolism of OA.更多还原