使用糖蛋白质组学分析与 2D-LC-MALDI 系统联用，寻找膝关节骨关节炎进展的潜在血浆生物标志物。

Potential plasma biomarkers for progression of knee osteoarthritis using glycoproteomic analysis coupled with a 2D-LC-MALDI system.

机构信息

Department of Pathomechanisms, Clinical Research Center, National Hospital Organization Sagamihara Hospital, Sagamihara, Kanagawa, 228-8522, Japan.

出版信息

Proteome Sci. 2012 Jun 6;10(1):36. doi: 10.1186/1477-5956-10-36.

DOI:10.1186/1477-5956-10-36

PMID:22672759

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3514375/

Abstract

BACKGROUND

Although osteoarthritis (OA) is a highly prevalent joint disease, to date, no reliable biomarkers have been found for the disease. In this study, we attempted to identify factors the amounts of which significantly change in association with the progression of knee OA.

METHODS

A total of 68 subjects with primary knee OA were enrolled in the study. These subjects were followed up over an 18-month period, and plasma and serum samples were obtained together with knee radiographs every 6 months, i.e., 0, 6, 12 and 18 months after the enrollment. Progressors and non-progressors were determined from the changes on radiographs, and plasma samples from those subjects were subjected to N-glycoproteomic 2D-LC-MALDI analysis. MS peaks were identified, and intensities for respective peaks were compared between the progressors and non-progressors to find the peak intensities of which differed significantly between the two groups of subjects. Proteins represented by the chosen peaks were identified by MS/MS analysis. Expression of the identified proteins was evaluated in synovial tissues from 10 OA knee joints by in situ hybridization, western blotting analysis and ELISA.

RESULTS

Among the subjects involved in the study, 3 subjects were determined to be progressors, and 6 plasma and serum samples from these subjects were subjected to the analysis together with another 6 samples from the non-progressors. More than 3000 MS peaks were identified by N-glycoproteomic 2D-LC-MALDI analysis. Among them, 4 peaks were found to have significantly different peak intensities between the progressors and non-progressors. MS/MS analysis revealed that these peaks represented clusterin, hemopexin, alpha-1 acid glycoprotein-2, and macrophage stimulating protein, respectively. The expression of these genes in OA synovium was confirmed by in situ hybridization, and for clusterin and hemopexin, by western blotting analysis and ELISA as well.

CONCLUSIONS

In this study, 4 potential biomarkers were identified as potential prognostic markers for knee OA through N-glycoproteomic analysis. To the best of our knowledge, this is the first report for the use of glycoproteomic technology in exploring potential biomarkers for knee OA.

摘要

背景

尽管骨关节炎（OA）是一种高发的关节疾病，但迄今为止，尚未发现该病的可靠生物标志物。在这项研究中，我们试图确定与膝骨关节炎进展相关的大量变化的因素。

方法

共有 68 名原发性膝骨关节炎患者参与了本研究。这些患者在 18 个月的随访中，每 6 个月（即入组后 0、6、12 和 18 个月）采集血浆和血清样本并进行膝关节 X 线检查。根据 X 线片的变化确定进展者和非进展者，并对来自这些患者的血浆样本进行 N-糖蛋白组 2D-LC-MALDI 分析。鉴定 MS 峰，比较进展者和非进展者各自峰的强度，以找到两组患者之间差异显著的峰强度。通过 MS/MS 分析鉴定代表所选峰的蛋白质。通过原位杂交、western 印迹分析和 ELISA 评估 10 个 OA 膝关节滑膜组织中鉴定出的蛋白质的表达。

结果

在所涉及的研究对象中，3 名被确定为进展者，对来自这 3 名患者的 6 个血浆和血清样本以及另外 6 个非进展者样本进行了分析。通过 N-糖蛋白组 2D-LC-MALDI 分析鉴定出 3000 多个 MS 峰。其中，有 4 个峰在进展者和非进展者之间的峰强度差异显著。MS/MS 分析表明，这些峰分别代表簇蛋白、血红素结合蛋白、α-1 酸性糖蛋白-2 和巨噬细胞刺激蛋白。通过原位杂交证实了这些基因在 OA 滑膜中的表达，并且对于簇蛋白和血红素结合蛋白，通过 western 印迹分析和 ELISA 也证实了其表达。