探討類風濕性關節炎患者低密度脂蛋白特徵作為導致其心血管疾病之前驅研究

Investigate the characteristics of low-density lipoprotein from rheumatoid arthritis patients for a pilot study of cardiovascular complications

黃詩涵(Shih-Han Huang)

指導教授 ： 柯良胤

高雄醫學大學/健康科學院/醫學檢驗生物技術學系碩士班/碩士(2018年)

摘要

中文摘要
類風濕性關節炎(Rheumatoid arthritis; RA)是一種常見的慢性發炎自體免疫疾。其疾病特徵是持續的高度關節發炎反應，所影響的範圍遍及全身關節，並且隨著關節疾病的嚴重程度增加常伴隨有心血管疾病…等關節外(Extra-articular)症狀的出現，最終導致RA患者的死亡。RA患者心血管疾病的風險與肥胖、高血壓、糖尿病等傳統因素不同，已知自體抗體（即類風濕因子、抗核抗體）、細胞發炎因子和脂質異常…等，可能導致這些患者發生動脈粥樣硬化和併發其他心血管疾病。
由於近年來陰電性低密度脂蛋白(electronegative low-density lipoprotein; L5 LDL)在體外和體內之相關研究報導均表現出具有導致動脈粥樣硬化特性，因此我們假設：L5 LDL在RA患者中扮演重要因子會導致併發心血管疾病。在本研究中，我們注重於調查RA LDL的陰電性，蛋白質組成和脂質成分分析，期望作為探討致病機轉之前驅實驗。
本研究利用快速蛋白質液相層析法作為檢測LDL的陰電性。再者，我們使用超高效能液相層析法(ultra performance liquid chromatography; UPLC)和平行碰撞誘導解離質譜(data-independent parallel-fragmentation mass spectrometry; MSE)用於鑑定RA LDL的蛋白質組成和脂質組分。數據以Progenesis QI-P及 QI 軟體進行統計分析。
與健康人(n=25)相比，RA患者(n=51) L5 LDL所佔體內total LDL的比例明顯增加(分別為0.39±0.48％ vs. 1.81±2.25％；P <0.01)。蛋白質體學的研究結果顯示：RA患者中apolipoprotein A-I (apoA-I), apolipoprotein A-II (apoA-II) ...脂蛋白含量增加。另外脂質成份分析結果顯示：RA LDL的溶血磷脂酰膽鹼(lysophosphatidylcholine; LPC)相對濃度3倍增加(P<0.001)；但神經酰胺和鞘氨醇(C17鹼基)在RA LDL中顯著減少(P<0.01)。
總之，RA患者LDL陰電性增加(electronegative LDL; L5)與LPC含量增加(LDL-LPC)可能是RA患者心血管疾病的病因。未來需要再進一步透過細胞的研究，來驗證L5 LDL與LPC在RA患者心血管疾病中的作用機制 。

關鍵字

類風濕性關節炎 ； 陰電性低密度脂蛋白 ； 心血管疾病

並列摘要

Abstract
Introduction: Rheumatoid arthritis (RA) is a common chronic inflammatory autoimmune disease characterized by a persistently high degree of joint inflammatory response. The severity of joint diseases is usually accompanied by the appearance of extra-articular symptoms such as cardiovascular diseases, leading to the death of RA patients. The risks of cardiovascular diseases in RA patients are different from traditional factors such as obesity, hypertension, diabetes… and so on. Autoantibodies (i.e. rheumatoid factor, anti-nuclear antibody), cytokines and lipid abnormalities may contribute to atherosclerosis and other cardiovascular diseases in these patients. However, there are some controversial reports regarding to the use of anti-inflammatory therapy. Detailed mechanisms for cardiovascular diseases in RA patients are still not clear.
Specific aims: Because electronegative low-density lipoprotein (L5 LDL) shows atherogenic properties in vitro and in vivo, we therefore hypothesize that L5 LDL plays an important role for cardiovascular diseases in RA patients. In this study, we focused on examining the electronegativity, of LDL protein composition and lipid components of RA LDL. We aim to investigate the difference of RA LDL. We believe this pilot study will contribute to the pathogenesis research of CVD in RA LDL.
Materials and Methods: Fast protein liquid chromatography was used to examine electronegativity of LDL. Ultra-performance liquid chromatography (UPLC) and data-independent parallel-fragmentation mass spectrometry (MSE) were used to identify protein composition and lipid components of RA LDL. Data was analyzed by Progenesis QI-P and QI software.
Results: L5 LDL was increased in RA patients (1.81±2.25 %) in comparison to healthy individuals (0.39±0.48%; P<0.01 respectively). ApoAI and apoAII were increased in RA patients. LPCs were significantly increased (3X increased) in RA LDL, but ceramide and sphinganine (C17 base) were significantly decreased.
Conclusion: L5 LDL and LDL-LPCs increased in RA patients may be the etiology of cardiovascular disease in RA. Further cell-based studies are needed to explore the mechanism and the role of L5 LDL and LDL-LPCs for cardiovascular diseases in RA patients.

並列關鍵字

Rheumatoid arthritis ； Electronegative low-density lipoprotein ； Cardiovascular disease

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