강직성척추염 환자의 활액에서 발현하는 특이 단백질 연구

Abstract

The synovial fluid is a lubricant, composed of hyaluronic acid, inflammatory cells and proteins released from synovial fibroblasts, synovial membrane and inflammatory cells. The development of peripheral arthritis in ankylosing spondylitis develops early in the disease course is a predictor of a more aggressive disease. The objective of this study was to identify differentially expressed protein mediators in synovial fluid of ankylosing spondylitis. A Total of 40 synovial fluid samples from 10 ankylosing spondylitis and each 10 controls (osteoarthritis, rheumatoid arthritis, gouty arthritis) were collected. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) were carried out to identify differentially expressed proteins based on the ratios of the extracted ion current of each protein between the four groups. 8 proteins showed 1.5 fold change, 7 of which were verified with the exception of the abundant protein Haptoglobin (HP). Matrix metalloproteinase-3 (MMP3) was used as a positive control, and the remaining 6 proteins were subjected to western blot analysis. 8 proteins were found to be more than 1.5-fold differentially expressed in synovial fluid of ankylosing spondylitis patients compared to control groups. Proteins such as HP, MMP3, Complement factor H-related protein 5 (CFHR5), Complement component C9 (C9), Mannose-binding lectin2 (MBL2), Complement C4-A (C4A), Serum amyloid P-component (APCS) and Matrix metalloproteinase-1 were found to be upregulated in the synovial fluid of ankylosing spondylitis patients. CFHR5 and C9 were reported in previous studies with ankylosing spondylitis serum. APCS was reported in synovial fluid as well as serum. However, C4A and MBL2 were newly discovered through this analysis. The study verified the unique expression level of C9 and CFHR5 in ankylosing spondylitis sample, using western blot analysis, as compared to the other three diseases. Quantitatively proteomic profiling of the respective synovial fluid sample from 4 diseases, i.e., ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, and gouty arthritis, by LC-MS/MS, was performed. The systematic comparative proteomic analysis of the four groups together was carried out for the first time, leading to several differentially expressed proteins in ankylosing spondylitis. Among them, C9 and CFHR5 were expected, whose expression levels were confirmed by western blot analysis and could be potential biomarker for ankylosing spondylitis.

활액은 윤활제로서, 활막 및 염증 세포로부터 방출되는 단백질로 구성된다. 강직성 척추염에서 말초 관절염은 질병 초기에 흔히 나타나며, 질병 활성도가 높다. 이 연구의 목표는 강직성척추염의 활액에서 차별적으로 발현된 단백질을 확인하는 것이다. 강직성척추염 10개와 각 10개씩의 비교군(류마티스관절염, 통풍관절염, 퇴행성관절염)으로, 총 40개의 활액 샘플을 수집했다. Liquid chromatography와 tandem mass spectrometry(LC-MS/MS)를 사용하여 4가지 그룹 사이의 추출 된 각 이온 전류 비율에 따라 차별적으로 발현된 단백질을 확인했다. Matrix metalloproteinase-3(MMP3)을 양성 대조군으로 사용하고, 많은 양의 단백질인 Haptoglobin(HP)을 제외한 나머지 6개의 단백질을 검증을 위한 Western blot 분석에 사용하였다. 8개의 단백질이 강직성척추염에서 증가되어 있었고, HP, MMP3, Complement factor H-related protein 5(CFHR5), Complement component C9(C9), Mannose-binding lectin2(MBL2), Complement C4-A(C4A), Serum amyloid P-component(APCS) 그리고 Matrix metalloproteinase-1이었다. 그 중, CFHR5와 C9은 강직성척추염의 혈청에서 보고된 바가 있으며, APCS는 혈청 뿐 아니라 활액에서도 보고가 되었다. C4A와 MBL2는 이번 분석을 통해 새롭게 발견된 단백질이다. 우리는 3가지 질병 대조군과 비교하여 Western blot 분석을 실시하였고, 강직성척추염 샘플에서 C9, CFHR5가 상향조절 된 것을 확인하였다. 이 연구는 관절염의 주요 4가지 질환을 가진 환자들로 부터 활액 샘플을 채취하여 LC-MS/MS 분석을 수행하였다. 네 그룹의 체계적인 단백체 분석은 처음으로 수행된 것이며, 강직성척추염에서 특별히 높은 발현을 보이는 8가지 단백질을 발견하였다. 그 중 Western blot 분석을 통해 C9, CFHR5를 검증했다. 이 두 단백질이 강직성척추염의 잠재적인 바이오마커가 될 것으로 기대한다.