膝滑液纤维连接蛋白水平与软骨损伤的相关性研究

A correlative study between synovial fibronectin and cartilage damage in the knee

[目的]观察膝软骨损伤滑液蛋白含量的变化,探索纤维连接蛋白(fibronectin,FN)是否可以作为一种可靠的生物学标记物来反映软骨损害的程度。[方法]131例软骨损害行手术治疗患者纳入本研究,术中全面评估软骨情况,按Outerbridge最高评级确定患者软骨损伤程度。取关节滑液进行SDS-凝胶电泳、考马斯蓝染色,识别有差异的条带进行质谱分析,分析得到的有表达差异的蛋白通过蛋白印迹实验来确定,进一步使用ELISA来验证。将软骨Outerbridge评级与滑液中筛选出的蛋白浓度对比,分析此蛋白与软骨损伤程度(Outerbridge最高评级)的相关性。[结果]SDS-PAGE电泳、考马斯蓝染色,比较后发现OA患者滑液中有一条约为220 KDa的深蓝色条带,切下进行质谱分析发现条带中含有大量FN的肽段。蛋白印迹检测发现FN在Outerbridge Ⅳ级滑液中的表达水平明显高于0级患者滑液中的水平。ELISA检测滑液FN水平按Outerbridge分级顺序如下:0级(87.07±5.65)μg/ml、Ⅰ级(102.75±5.88)μg/ml、Ⅱ级(201.90±12.23)μg/ml,Ⅲ级(287.13±12.51)μg/ml,Ⅳ级(303.63±7.32)μg/ml。FN的变化与软骨损伤的严重程度呈正相关(r=0.873,P<0.01)。[结论]在膝滑液中FN水平可以作为一种早期的生物标记来反映软骨损伤情况,并可以在一定程度上反映出膝关节软骨损伤的程度。

[Objective] To observe the changes of synovial protein content in knee cartilage damage, and to explore whether fibronectin（FN） can be used as a reliable biomarker for the extent damage. [Methods] This study included 131 patients who suffered from knee cartilage damage and underwent surgical treatment. During operation, the articular cartilage was thoroughly evaluated and the extent of cartilage damage was determined by the maximum Outerbridge grade. After synovial fluids were assessed with SDS-gel electrophoresis and Coomassie blue staining, different proteins in the bands were identified using mass spectrometry analysis, and further confirmed by western blotting. Subsequently, the protein concentrations in the synovial fluid were detected by Elisa. The correlation between the protein concentrations and the extent of cartilage damaged（the maximum Outerbridge grade） was statistically analyzed. [Results] A deep blue band about 220 KDa was found after Coomassie blue staining, and analyzed by Mass spectrometry. It was found that the band contained a large amount of fibronectin（FN） peptides. Western blotting showed that the FN levels in the joints ranked as Outerbridge Grade Ⅳ were significantly higher than the joints ranked as Grade 0. Regarding to the Elisa assay, the FN level proved（87.07±5.65） μg/ml in Grade 0,（102.75±5.88） μg/ml in Grade Ⅰ,（201.90±12.23） μg/ml in Grade Ⅱ,（287.13±12.51） μg/ml in Grade Ⅲ and（303.63±7.32） μg/ml in Grade Ⅳ in order of Outerbridge classification, there was a significantly positive correlation between the FN level in the synovial fluids and the severity of cartilage damage（r=0.873, P〈0.01）. [Conclusion] The level of FN in knee synovial fluid might be used as an early biomarker of cartilage damage, and reflect the severity of cartilage damage of the knee in some extent.