心肌缺血血瘀证小型猪模型差异蛋白质组学研究

Quantitative and comparative proteomics analysis of chronic myocardial ischemia pig models with blood stasis syndrome

目的:利用基于荧光差异凝胶电泳(DIGE)技术的差异蛋白质组学的方法筛选心肌缺血血瘀证小型猪血浆差异表达的蛋白,以期发现心肌缺血血瘀证特征性的差异蛋白质或蛋白质群。方法:对模型动物施行冠脉Ameroid环缩术,通过动态观察,综合评价,明确术后4周动物表现为冠心病(心肌缺血)血瘀证稳定的时间点。4周时前腔静脉取血,枸橼酸钠抗凝并加入蛋白酶抑制剂,血样处理后进行荧光双向差异凝胶电泳(2D-DIGE),运用Decyder软件分析得到差异蛋白点,进行MALDI-TOF/TOF分析,获取蛋白样品的肽质量指纹图。结果:共筛选出31个差异表达蛋白质点,其中17个蛋白质点在4周模型组中下调,14个蛋白点上调。对其中15个蛋白点采用质谱技术成功鉴定。白蛋白、血红素蛋白、烟酸受体在4周模型组中低表达,CH4 and secrete domain of Swine IgM、mutated immunoglobulin heavy chain、肌球蛋白、磷脂酶C、白细胞抗原相关酪蛋白磷酸酶相关蛋白、磷酸核糖焦磷酸相关蛋白-1、Ig gamma4 chain constant region在4周模型组中高表达。结论:初步发现白蛋白、碱性磷酸酶、肌球蛋白、白细胞抗原相关酪蛋白磷酸酶相关蛋白、磷酸核糖焦磷酸合成酶相关蛋白1、血红素蛋白、烟酸受体、CH4 and secrete domain of Swine IgM、mutated immunoglobulin heavy chain、磷脂酶C可能与心肌缺血血瘀证相关,有待于进一步的验证及机制研究。

Objective：To identify specific protein markers for chronic myocardial ischemia with blood stasis syndrome. Methods： 6 healthy pigs were included in the experiment. After thoracotomy and pericardiectomy, ameroid constrictor was implanted around the left anterior descending artery. After operation, observation was taken continuously. Then the chronic myocardial ischemia was evaluated by coronary arteriography and electrocardiogram. At the same time, blood stasis syndrome was diagnosed according to the standard recommended by Chinese Association of Integrated Traditional and Western Medicine. It is proved that 4th week after operation is the point to stable stage of blood stasis syndrome. The plasma samples of model group and control group were examined by the fluorescence differential in-gel electrophoresis （2D-DIGE） technique. Protein spots detected differential with statistical significance were identified by MALDI-TOF/TOF. Results：Intensity changes of 31 spots were detected with statistical significance, l 7 protein spots of them were down-regulated, 14 were up-regulated in 4th week models. And 15 of them were identified by MALDI-TOFfrOF successfully. Several proteins were down-regulated such as albumin, hemopexin, nicotinic acid receptor. CH4 and secrete domain of Swine IgM, mutated immunoglobulin heavy chain, myosin heavy chain, PLC, PRPS-associated protein l,Ig gamma 4 chain constant region were up-regulated. Conclusion： It is proved that albumin, alkaline phosphatase, myosin heavy chain, PLC,PRPS-associated protein 1,hemopexin,nicotinic acid receptor are possibly the specific proteins for chronic myocardial ischemia with blood stasis syndrome.But the conclusion needs further validation and mechanism studies.