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目的本研究利用差异蛋白质组的方法筛选心力衰竭大鼠线粒体差异表达的蛋白,以期发现心力衰竭发生的新机制或新的治疗靶点。方法对大鼠行左冠状动脉前降支结扎,术后4周进行超声和血流动力学检测,成功建立心力衰竭大鼠模型。实验动物分为心力衰竭组和对照组,每组9只。采用差速离心法分别分离纯化左心室。利用双向凝胶电泳技术显示差异蛋白的表达谱。结果差异比较结果显示,有15个点表达的差异有统计学意义,经胶内酶解后,对上述差异表达的蛋白点进行胶内酶解后用LC／MS进行分析,然后利用串级质谱数据通过蛋白数据库检索对蛋白进行鉴定。共有8条蛋白在心力衰竭时显著下调(5倍以上),包括乙醛脱氢酶2 (ALDH2)、肌球蛋白轻链(myosin, light polypeptide 3)、顺乌头酸酶(Aconitase 2)、黄素蛋白(electron-transfer-flavoprotein, beta polypeptide)等;另有7条蛋白在心力衰竭时显著上调(5倍以上),包括波形蛋白(vimentin)、微管蛋白(tubulin,beta 5)、二氢硫辛酰胺S-乙酰转移酶(dihydrolipoamide S-acetyltransferase)等。鉴于ALDH2参与话性氧代谢的文献报道,对其作进一步鉴定。又运用反转录-聚合酶链反应和Western印迹法检测心肌梗死后不同时期心肌ALDH2的表达情况,发现在心肌梗死后3、7、14和28 d心肌ALDH2的mRNA和蛋白表达呈逐渐下降趋势。结论利用蛋白质组技术得到在大鼠心力衰竭心肌中表达显著改变的15条蛋白,并对1条在心力衰竭心肌中显著下调的蛋白ALDH2进行鉴定,为下一步的差异蛋白功能研究奠定了基础。
Objective In this study, differential proteomic methods were used to screen differentially expressed proteins of mitochondria in heart failure rats in order to find out a new mechanism of heart failure or a new therapeutic target. Methods The left anterior descending coronary artery was ligated in rats. Ultrasound and hemodynamics were performed 4 weeks after the operation. The rat model of heart failure was successfully established. Experimental animals were divided into heart failure group and control group, 9 in each group. Differential centrifugation were used to separate and purify the left ventricle. Two-dimensional gel electrophoresis technique was used to show the differential expression profiles. Results Differences in the results showed that there were 15 points of difference was statistically significant, after gel in solution, the above differentially expressed protein spots were gel-digested with LC / MS analysis, and then use the tandem mass spectrometry Data were verified by protein database search. A total of 8 proteins were significantly down-regulated (over 5-fold) in heart failure and included ALDH2, myosin light polypeptide 3, Aconitase 2, The other seven proteins were significantly upregulated (more than 5-fold) in heart failure, including vimentin, tubulin, beta 5, Hydrolipamide S-acetyltransferase and the like. In view of ALDH2 involved in the literature of the oxygen metabolism, it is further identified. Reverse transcription - polymerase chain reaction and Western blotting were also used to detect the expression of ALDH2 in myocardium at different stages after myocardial infarction. The mRNA and protein expression of ALDH2 in myocardium decreased gradually at 3, 7, 14 and 28 days after myocardial infarction . CONCLUSION: Fifteen proteins with significant changes in myocardial tissue of rats with heart failure were obtained by proteomic technique and identified a protein ALDH2 significantly down-regulated in heart failure, which laid the foundation for the further study of differential protein function.