脂联素蛋白在膝骨关节炎血清中的表达

Study of adiponectin as knee osteoarthritis serum biomarkers

目的通过对膝骨关节炎(KOA)Kellgren-Lawrence分级(K-L分级)的各阶段的血清样本进行同位素标记相对和绝对定量(i TRAQ)蛋白组学分析,查找正常组与KOA组的血清差异蛋白。方法收集符合美国风湿病学院诊断标准KOA患者60例,再参照K-L分级标准,分为K-L 0、Ⅱ、Ⅳ级3个亚组进行对比,每个亚组单纯随机抽样选取10例男性与10例女性。去除血清高丰度蛋白,进行稳定同位素116、117、118的i TRAQ标记、反相色谱柱分离、质谱检测,采用Swissport数据库检索蛋白峰图,并行生物信息学分析。采用Western blot技术对代表性血清差异蛋白行表达验证。并采用Image J软件计算条带的曝光值,分析差异蛋白组间差异。结果通过对不同样品的i TRAQ肽段实验标记、Q-star质谱鉴定和Mascot蛋白搜索软件搜库,共筛选出有意义差异蛋白169个。通过K-L 0级组与K-LⅡ级、Ⅳ级组对比,共筛选出有意义差异蛋白89个,其中上调的差异蛋白有75个,下调的差异蛋白有14个。通过Western blot验证结果显示,与正常对照组相比,脂联素蛋白在KOA患者血清中表达量上调(F=10.06,P<0.05),C反应蛋白表达下调(F=6.26,P<0.05)。结论本实验通过i TRAQ定量蛋白组学分析和Western blot验证发现脂联素蛋白在KOA患者的血清中的上调表达。脂联素蛋白可能是KOA发生发展过程中的生物标志物,为KOA发生发展机制及血清标志物的研究提供了新的线索。

Objective To screen serum differentially expressed proteins in knee osteoarthritis （KOA） patients and search biomarkers for KOA diagnosis by isobaric tag for relative and absolute quantitation. Methods Sixty serum samples from the patients with knee osteoarthritis were enrolled, and according to Kellgren-Lawrence （K-L） grading, the patients were divided into K-L 0, 11 and Ivgroups. High-abundance proteins in serum were removed, isobaric tags for relative and absolute quantitation （iTRAQ） coupled with two-dimensional liquid chromatography/tandem mass spectrometry （2D-LC-MS/ MS） were used to analyze and identify differentially expressed serum proteins among the three groups. Bioinformatics was used to analyze the identified differentially expressed proteins, and the expression of representative differential proteins was verified by Western blot. The differences between protein groups were analysed by using the Image J software. Results The iTRAQ coupled with 2D-LC-MS/MS analyses led to the identification of 169 proteins. Among the K-L 0,Ⅱ and IV, 89 proteins were differentially expressed including 12 up-regulated ones and five down-regulated ones in the OA patients. Western blot results showed that compared with the normal controls, the expression of representative differential protein （adiponection monoclonal antibody, ADIPOQ） in the serum of OA patients was significantly up-regulated and C-reactive protein （CRP） in the serum of OA patients was significantly down-regulated （ P 〈 0.05 ）. Conclusions Adiponectin is validated to be up-regulated in KOA through iTRAQ quantitative proteomics analysis and Western blot. The resuhs suggest that ADIPOQ is potential diagnostic or prognosis biomarker for KOA, and it may play an important role in development of KOA.