双向荧光差异凝胶电泳技术在心脏病蛋白质组学研究中的应用

Application of 2D Differential-in-gel Electrophoresis for Analysis of Comparative Proteomics of Heart Failure Due to Arrhythmogenic Right Ventricular Cardiomyopathy

目的:应用双向荧光差异凝胶电泳技术筛选与致心律失常性右心室心肌病引起的与心力衰竭密切相关的蛋白质标志物。方法:从本院的心脏病组织库中挑选6例病理诊断明确和各方面资料比较齐全的致心律失常性右心室心肌病引起心力衰竭的左心室心肌标本(来源于心脏移植的受体),以年龄、性别和种族等因素相匹配的因非心脏病死亡的6例正常心脏的左心室心肌作为本实验的对照,进行比较蛋白质组学研究,筛选在致心律失常性右心室心肌病引起的心力衰竭中差异表达的蛋白质,即蛋白质标志物,并应用免疫组织化学和免疫印迹杂交方法检验差异蛋白质的可靠性。结果:经二次重复实验共筛选出5个差异表达的蛋白质,其中有3个在致心律失常性右心室心肌病引起的心力衰竭中表达升高,而另有2个表达降低。经生物信息学分析,这5个差异蛋白质主要为心肌细胞的骨架蛋白或参与能量代谢和应急保护反应的蛋白质。其中的一个差异表达蛋白质——细胞骨架蛋白 Desmin 经免疫组织化学和免疫印迹杂交方法得到了进一步验证。结论:本研究应用双向荧光差异凝胶电泳技术筛选获得5个与致心律失常性右心室心肌病引起的心力衰竭密切相关的蛋白质标志物,这些标志物可能与心力衰竭发生的分子机制相关,并可能用于疾病的分层、判断预后及指导个性化治疗。

Objective ：To sieve the protein markers associated with heart failure derived from arrhythmogenic right ventricular cardio myopathy（ ARVC ） by 2D differential-in-gel electrophoresis （2D-DIGE）. Methods：We performed 2D-DIGE analysis using individual left ventricular myocardial tissue from 6 patients with heart fai lure due to ARVC undergoing heart transplantation. In control experiment, we chose 6 non-failing subjects for left ventricular myocardial tissue with the matched age, sex and race to screen the differentially expressed proteins as potential protein markers associated with heart failure derived from ARVC. The results were further confirmed by immunohistochemistry and Western blot analysis. Results：We identified 5 differentially expressed proteins by twice repeated experiments, three of which showed increased protein expression and two decreased in ARVC failing heart compared to non-failing control. Based on 2-DE gel protein databases of human cardiac protein that have been established, the differentially expressed proteins were classified into three functional classes of cytoskeleton,energy production and stress response. Desmin,one of the highly expressed proteins,was further vaidated by immunohistochemistry and Western blot analysis. Conclusions：We first identified those five differentially expressed proteins, potentially as protein markers, associated with heart failure due to ARVC, which might be related to the molecular mechanism of heart failure and might be useful to detect the risk stratification, assess the prognosis, and guide the management of heart failure.