Progress of proteomics technology and its application in osteoarthritis

doi:10.3969/j.issn.2095-4344.2014.24.023

Abstract

Abstract:

BACKGROUND: Proteomics research has thoroughly reformed the way to study the diseases, and provides the understanding of the mechanism of osteoarthritis pathological physiology.
OBJECTIVE: To focus on the research progress of proteomics technology in osteoarthritis early diagnosis, therapeutic effect and pathogenesis molecular markers.
METHODS: Researches on proteomics technology and osteoarthritis published in journals were identified manually and on-line retrieved by using SinoMed, Chongqing VIP database, Wanfang Data and CNKI database. Those researches reported in English journals were identified using MEDLINE, Elsevier: ScienceDirect, and BIOSIS Previews database. Selected studies should describe a related and reliable study defined by strict screening and diagnostic criteria.
RESULTS AND CONCLUSION: Proteomics technology for the osteoarthritis in early diagnosis, treatment effect and pathogenesis molecular markers has achieved a certain progress. Research showed that proteomics has great application value in screening osteoarthritis molecular markers, and has the characteristics of high flux and high speed compared with single protein identification, so it plays an important role in the diagnosis of osteoarthritis and drug target identification. Proteome database is a sign of proteome research level. The development of the bioinformatics has provided a more convenient and effective computer analysis software for proteomic study.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: osteoarthritis, proteomics, mass spectrometry

CLC Number:

R318

Pan Ding, Lei Guang-hua . Progress of proteomics technology and its application in osteoarthritis[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(24): 3912-3918.

Figures/Tables 1

2.1 蛋白质组学概述蛋白质组是指由一个基因组，或一个细胞、组织表达的所有蛋白质[19]。蛋白质组的概念与基因组的概念有许多差别，它随着组织结构、甚至环境状态的不同而改变。一个基因在转录后可以多种mRNA形式进行剪接，并且同一个蛋白质也可能以许多形式进行翻译后的修饰。因此一个蛋白质组并不是一个基因组的直接产物，蛋白质组中蛋白质的数目可以超过基因组的数目[20]。蛋白质组学研究集中于动态地描述基因的各种调节，对基因的表达在蛋白质水平上进行定量地测定，鉴定药物和疾病对人体组织的影响，以及尝试解释基因表达调控的机制[21-22]。蛋白质组学研究是基因产物图谱和蛋白质(多肽)图谱技术的一种延伸，基因产物图谱依靠多种物理化学方式分离后的分析，如质谱技术和氨基酸组分分析等[23]；而蛋白质(多肽)图谱则依靠双向凝胶电泳技术和进一步的图象分析。国际上蛋白质组学研究已经取得了一定的进展，不论是在基础理论方面还是技术方法上，都在不断完善和进步。目前已经建立了多种蛋白质组学数据库。1996年，澳大利亚建立了世界上第一个蛋白质组研究中心：Australia Proteome Analysis Facility (APAF) [24]。2001年4月，在美国成立了国际人类蛋白质组研究组织(Human Proteome Organization, HUPO) [25]，随后欧洲、亚太地区都相继成立了区域性蛋白质组研究组织，尝试通过合作的方式，融合各方面的力量，完成人类蛋白质组计划(Human Proteome Project) [26]。 2.2 蛋白质组学研究内容 2.2.1 蛋白质鉴定利用1D电泳和2D电泳并结合免疫组织化学和Western blot等技术，或者利用免疫共沉淀、蛋白质芯片及抗体芯片等技术对组织中的蛋白质进行筛查和鉴定[27]。 2.2.2 蛋白质翻译后修饰蛋白质在经过mRNA表达后，还需经历复杂的翻译后修饰如糖基化，磷酸化和酶原激活等。翻译后修饰是调节蛋白质功能的一种重要途径，因此对蛋白质翻译后修饰的研究对阐明蛋白质的功能具有重要作用[28]。 2.2.3 蛋白质功能分析主要包扩酶活性的分析和鉴定对应酶的底物，以及细胞因子功能网络或信号转导通路的分析。利用的技术主要包括基因敲除技术和反义表达技术。此外研究表达蛋白在细胞中的定位在一定程度上也有益于蛋白质的功能分析，比如Clontech公司研发的荧光蛋白表达系统就是研究细胞内蛋白质定位的一种工具[29]。 2.2.4 靶蛋白的寻找蛋白质是很多药物的单一或复合组分，而很多药物的受体或靶分子也是蛋白质。除了直接作用于靶蛋白外，也可以通过对靶蛋白的研究来设计出干预蛋白质-蛋白质相互作用的药物分子[30]。了解和探索人类不同生长、发育期和不同生理、病理条件下及不同细胞类型的基因表达特点，对于基础和临床医学病理机制研究具有特别重要的意义。通过相关蛋白质组学研究可以找到直接与特定的与生理病理状态相关的蛋白质分子，为进一步设计作用于靶蛋白的药物奠定基础[31]。 2.3 蛋白质组学技术分类蛋白质组学技术的发展已经成为现代生物技术快速发展的重要支撑，在近些年骨代谢和骨生物学研究方向取得了关键性的突破，尤其是在骨组织相关细胞的胞内信号传导方面取得了一定的进展[32]。 2.3.1 双向凝胶电泳双向凝胶电泳的原理是第一向基于蛋白质的不同等电点采用等电聚焦分离，第二向则按不同的分子量采用SDS-PAGE分离，把复杂蛋白混合物中的总蛋白在二维平面上分离。由于双向电泳技术在蛋白质组与医学研究中所处的重要位置，它可用于蛋白质转录及转录后修饰研究、蛋白质组的比较和蛋白质间的相互作用、细胞分化凋亡研究、致病机制及耐药机制的研究、蛋白纯化等许多方面。近年来经过多方面改进已成为研究蛋白质组的最有使用价值的核心方法[33]。 2.3.2 等电聚焦等电聚焦是一种利用有pH梯度的介质分离不同等电点蛋白质的电泳技术。等电聚焦凝胶电泳依据蛋白质分子的静电荷或等电点进行分离，蛋白质分子在含有载体两性电解质形成的一个连续而稳定的线性pH梯度中电泳。蛋白质分子在偏离其等电点的pH条件下带有电荷，因此可以在电场中移动；当蛋白质迁移至其等电点位置时，其静电荷数为零，在电场中不再移动，据此原理将蛋白质分离[34]。 2.3.3 生物质谱生物质谱技术是蛋白质组学研究中最重要的鉴定技术，其基本原理是样品分子离子化后，根据不同离子之间的荷质比(M/E)的差异来分离并确定分子量。对于经过双向电泳分离的目标蛋白质用胰蛋白酶酶解(水解Lys或Arg的-C端形成的肽键)成肽段，对这些肽段用质谱进行鉴定与分析[35]。目前常用的质谱技术包括以下两种：基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF- MS)和电喷雾质谱(ESI- MS)(图1)。"

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