同位素相对标记技术（iTRAQ）结合双向液相色谱与质谱联用技术应用于膝骨性关节炎血清差异蛋白的实验研究

发布时间：2018-07-26 12:50

【摘要】：膝关节骨性关节炎(Knee osteoarthritis, KOA)是最常见的关节疾患之一,主要表现为关节缓慢发展的疼痛、僵硬、肿大,伴关节功能障碍,甚至发生残疾。随着老龄化社会的到来,KOA对人类的健康和生存质量影响增大,本病的发病率日趋升高,对其的研究已成为医学领域中的重要课题。国内外公认的是膝关节骨关节炎是一种长期的、隐匿的慢性疾病。诊断通常是基于临床症状和影像学变化。然而X线灵敏度、精确度相对比较差,目前临床上的诊断手段不允许0A的早期诊断或关节损伤进展的评估。因此,寻找膝关节骨性关节炎的生物标志物作为0A早期的诊断标准是目前研究的一个热门方向。蛋白质组学能通过对全体蛋白质表达的规模化分析,研究生物过程相关蛋白质的动态表达和功能的改变；对于寻找疾病诊断的特异性标志物以及对疾病的发病机理的研究来说,是一种有效的高通量的研究方法,可以获得传统手段无法得到的蛋白标志物,目前已经成为寻找新的肿瘤蛋白标志物的主要方法。本研究我们首先运用蛋白质组学研究方法及手段,通过对膝骨性关节炎K-LO、Ⅱ、Ⅳ级的3个亚组的血清进行差异蛋白质组学分析,筛选出了一些有潜在诊断价值的差异蛋白质,并对其中部分差异蛋白质进行了验证和进一步分析。目的：通过对膝骨性关节炎K/L分级的各阶段的血清样本进行ITRAQ定量蛋白组学分析检测进行差异蛋白的筛选,以发现膝骨性关节炎各个时期的的潜在分子标志物。方法：收集膝关节骨性关节炎患者60例,参照Kellgren-Lawrence(K-L)分级标准,分为K-L 0、 Ⅱ、Ⅳ级的亚组,每个亚组随机选取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iTRAQ标记、反相色谱柱分离、质谱检测及Swissport数据库检索；再利用生物信息学软件进行分析。对的部分差异蛋白进行Western Blot验证。结果：通过对不同样品的iTRAQ肽段实验标记、Q-star质谱鉴定和MASCOT搜库,共筛选出有意义差异蛋白169个,K-L 0级与K-LⅣ级差异蛋白153个K-L 0级与K-L Ⅱ级差异蛋白153个；K-L II级与K-L IV级差异蛋白145个。Western Blot验证的ADIPOQ、CRP两个差异蛋白与实验iTRAQ定量蛋白组学分析检测的结果相符合。结论：1.应用iTRAQ技术可筛选出与膝关节骨性关节炎发生发展相关的多个差异蛋白。提示iTRAQ技术对于膝关节骨性关节炎蛋白质组学血清生物标记物的研究有很好的应用前景。2. Adiponectin可能是诊断膝关节骨性关节炎早期潜在的血清生物标志物。同时指出脂类代谢紊乱可能才是膝骨性关节炎发病的根本原因。
[Abstract]:Knee osteoarthritis (Knee osteoarthritis, KOA) is one of the most common joint diseases, which is characterized by slow development of joint pain, stiffness, swelling, joint dysfunction and even disability. With the arrival of an aging society, the impact of KOA on human health and quality of life is increasing, the incidence of this disease is increasing, and its research has become an important subject in the field of medicine. Knee osteoarthritis is recognized at home and abroad as a long-term, hidden chronic disease. Diagnosis is usually based on clinical symptoms and imaging changes. However, X-ray sensitivity and accuracy are relatively poor. The current clinical diagnostic methods do not allow the early diagnosis of 0A or the evaluation of the progression of joint injury. Therefore, looking for biomarkers of knee osteoarthritis as an early diagnostic criterion of 0 A is a hot topic. Proteomics can study the dynamic expression and functional changes of proteins associated with biological processes through large-scale analysis of the expression of all proteins; for searching for specific markers for disease diagnosis and for studying the pathogenesis of disease, It is an effective high-throughput research method, which can obtain protein markers that can not be obtained by traditional methods, and has become the main method to find new tumor protein markers. In this study, we first used proteomics research methods and methods to analyze the differential proteomics of three subgroups of knee osteoarthritis (K-LOO, 鈪，

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