【作者】 仇琪；

【导师】 王伟；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2010， 博士

【摘要】 目的：建立稳定、可重复并能用于中药药效评价的心肌缺血大鼠病证结合动物模型,并应用非标记定量蛋白质组学研究策略对病证结合模型动物证候生物标志分子进行研究。通过心肌缺血气虚血瘀证证候特征性蛋白质或蛋白质群的发现,探究“气虚-气虚血瘀-气虚”证候及证候转化过程中,心肌蛋白质表达的变化规律,为心肌缺血气虚血瘀证证候的生物学基础研究提供新的证据；为疾病证候的诊断提供新的标志物；为对证治疗提供新的靶标。通过对心肌缺血病证结合模型动物心肌蛋白质组的动态检测、鉴定和后续的生物信息学处理,将检出的蛋白质根据功能和表达水平发生改变的时间进行分类,为中药对证治疗策略的形成和治疗靶点的确定提供依据。方法：1.采用基于Shannon熵互信息的方法对证候相关理化指标进行文献研究,筛选出与缺血性心脏病证候关系最为密切的理化指标；2.采用左冠状动脉结扎法造成大鼠急性心肌缺血动物模型,运用病证结合的思路,对术后4、7、14、21、28、45、60d模型组和假手术组动物的宏观指标进行全面采集的同时进行理化指标跟踪检测,将宏观指标与理化指标相结合,对不同时间点模型动物的证候属性进行判定；3.根据各时间点模型动物心肌组织HE染色和细胞凋亡检测结果选择进行蛋白质组检测的目的心肌组织；4.采用非标记定量蛋白质组策略对术后4、14、28、45d,即两个气虚证时间点和两个气虚血瘀证时间点的模型组左心室梗死边缘区和假手术组左心室心肌样品进行蛋白质组分析检测；通过GO注释和KEGG信号通路注释对差异蛋白质进行生物学过程、细胞成分、分子功能分类和细胞信号通路注释,对差异蛋白的证候相关性进行初步阐释；5.采用Western-blot法对部分差异蛋白的动态表达情况进行验证。结果：1.现有文献中,有相关理化指标研究的证候要素包括气虚、血瘀、痰浊、阴虚、寒凝、阳虚、气滞等7个,涉及理化指标134个。其中,与气虚证最相关的理化指标为超声心动,血液流变学指标与血瘀证相关系数最高。2.术后4d,模型组大鼠被毛竖立,懒动,易激惹；与同时间点假手术组大鼠比较,安静状态下呼吸频率明显减慢,应激后呼吸频率显著增快,左心室射血分数显著降低,左室短轴缩短率降低,舌、耳廓、足底色度以及血液流变学指标无显著差异。术后7-28d,模型组大鼠被毛无光泽,活动度明显减低,对外界刺激反应迟钝；与假手术组大鼠比较,模型组大鼠安静状态下呼吸频率明显减慢,应激后呼吸频率显著增快,游泳力竭时间明显缩短,左心室射血分数和左室短轴缩短率均显著降低,舌、耳廓、足底色度值及血液流变学指标与假手术组大鼠相比存在显著差异；术后45-60d,模型组大鼠被毛无光泽,活动度明显降低,对外界刺激反应逐渐迟钝,挣扎无力；与假手术组比较安静状态下呼吸频率明显减慢,应激后呼吸频率显著增快,左心室射血分数和左室短轴缩短率进一步降低,舌、耳廓、足底色度值及血液流变学指标与假手术组大鼠相比无显著差异。结合临床气虚血瘀证诊断标准,对左冠结扎术后不同时间点模型动物证候属性进行判定,术后4d为气虚证,术后7-28d为气虚血瘀证,术后45-60d为气虚证。Lars回归显示动物各部位色度改变与红细胞压积和血小板聚集率改变有关。3.心肌组织HE染色和原位细胞凋亡检测可见,术后各时间,点模型组大鼠心肌梗死区以心肌坏死为主要改变,左心室梗死边缘区表现为炎细胞浸润、细胞凋亡、细胞肥大等一系列病理改变。4.对术后4个时间点模型组大鼠左心室梗死边缘区和同时间点假手术组大鼠左心室心肌进行非标记定量蛋白质组分析检测结果为,在8组样品中共鉴定到526个蛋白。其中467个蛋白在4个时间点均被鉴定到。以蛋白表达量比值0.8～1.2作为阈值对4个时间点的蛋白进行筛选,得到45个差异蛋白可能与“气虚证-气虚血瘀证-气虚证”演变过程相关。通过GO功能注释、模糊聚类和KEGG细胞信号通路注释发现,差异蛋白质主要包括细胞骨架蛋白、代谢相关酶类、氧化应激相关蛋白和物质转运相关蛋白。这些蛋白质在维持细胞结构,增强心肌收缩力、调节细胞能量代谢、诱导细胞凋亡以及细胞内信号转导等方面发挥着重要的作用。5.经Western-blot法对肌动蛋白、alphaB晶体蛋白、L-乳酸脱氢酶、热休克蛋白8表达的动态变化验证结果与蛋白质组检测分析结果基本一致。结论：1.通过左冠状动脉结扎法造成的大鼠急性心肌缺血模型动物在术后不同时间段内表现为不同的证候。该模型能够应用于冠状动脉病变导致的心肌缺血气虚证和气虚血瘀证的生物学基础和中药药理研究。2.心肌蛋白质组检测结果能够反映心肌缺血疾病的进展和证候演进情况,心肌蛋白质组动态变化与心肌缺血疾病证候的动态演变相关。更多还原

【Abstract】 Objective:To establish a stable and repeatable disease and syndrome combination animal model on myocardial infarction rats, and to quest the biomarkers through the application of label free quantitative proteomics strategy.To inquiry the variation of protein expression during the syndrome and syndrome transformation of "Qi deficiency-Qi deficiency and Blood stasis-Qi deficiency", in order to provide the new proof for the biological basis, the new biomarkers, and the new therapeutic target for the syndrome of Qi deficiency and Blood stasis after acute myocardial infarction.To check against the formation and to provide the basis to the determination of therapeutic targets of Chinese medicine, which based on the proteins cluster according to the expression changes resulted from the detection, identification, and the analysis by the bioinformatics technology on the myocardium proteomics of the model animals.Methods:1. The Data mining technology based on the Shannon entropy mutual information were。used to analysis the complicated correlations of the statistical distribution of coronary heart disease syndromes associated physical and chemical indexes.2. SD rats were subjected to either proximal LAD ligation or sham operation.20 rats (10 in each group) were subjected to forced swimming test (FST) to measure the exhaustive swimming time in every time point. Echocardiography was used to evaluate the cardiac function in rats at each time point, and the respiratory rate in both peaceful state and post-stress were counted. After the images of ear concha, foot plant and facies lingualis were took, the color indexes were analyzed by Photoshop software, and the hemorheology were measured at the same time. LARS was subjected to calculate the correlation between the color and hemorheology.3. Label-free nanoscale LC-MS quantitative proteomics was applied in this study to inquiry the variation of protein expression during the syndrome and syndrome transformation of "Qi deficiency-Qi deficiency and Blood stasis-Qi deficiency" on the myocardium of myocardial infarction model animals at 4th,14th,28th,45th after operation. Fuzzy Clustering, GOslimviewer, and KEGG signaling pathway was used to do the annotation of time course expression, molecular function, cellular component, biological process, and the signaling pathway of the differential proteins.4. Western blotting was used to corroborate the proteomic results. Results:1.7 in 13 syndrome factors including qi deficiency,blood stasis,turbid phlegm,yin deficiency,cold coagulation,yang deficiency,qi stagnation which have been most researched are involved in about 134 physical and chemical indexes mentioned in the literature.The top 10 physical and chemical indicators of each syndrome factors were obtained after analysis calculation.Echocardiography is the most relevant indicator of Qi deficiency,and hemorheology and blood stasis has the highest correlation.2.Compared to sham operation group,the respiratory rate in the peaceful state was slower,but faster post stress,and the cardiac function of rats is significant decreased in model group during the period from 4th day to 60th day.The exhaustive swimming time is much shorter,and both color and hemorheology indexes showed significant differences between the two groups during the period from 7th day to 28th after operation.LARS showed that there was significant correlation between color indexes and results of hematocrit and platelet aggregation rate.All the results shows that the myocardial infarction rats induced by LAD ligation have Qi-deficiency indications,can be diagnosed as the syndrome of Qi-deficiency at 4th day；have both Qi-deficiency and Blood stasis during the period of 7th day to 28th day after operation, can be diagnosed as Qi-deficiency and Blood stasis syndrome；and only have Qi-deficiency indications,can be diagnosed as Qi-deficiency syndrome during the period of 45th day to 60th day,after operation.3.HE staining and myocardium in situ cell death detection showed that myocardial necrosis is the major pathological change in the infarct area at different time points after myocardial infarction, and infarct border zone in left ventricular showed a series of pathological changes as inflammatory cell infiltration,apoptosis,and cell hypertrophy.4.The myocardium proteome analysis showed that samples were identified in 8 groups of 526 proteins.Among which 467 proteins in the four time points were identified.45 different proteins may be asso-iated with"Qi deficiency-Qi deficiency and Blood stasis-Qi deficiency" evolution-related.The Fuzzy clustering,GO functional annotation,and KEGG cell signaling pathway annotation showed these proteins include cytoskeletal proteins, metabolic enzymes,oxidative stress related proteins and ion channel protein.They might play important roles in maintain cell structure,increase cardiac contractility,regulate intracellular energy metabolism,induce apoptosis and intracellular signal transduction.5.The dynamic changes of the expression of actin, alphaB crystallin, L-lactate dehydrogenase,and heat shock protein 8 which was tested by the Western blotting assay was concordant with the label-free quantitative proteomic results.Conclusion:1.The rats underwent acute myocardial infarction caused by the left coronary artery ligation showed different syndromes in different stages after operation.The animal model can be used in the biological basis research and Pharmacological studies of Chinese medicine on Qi deficiency syndrome or Qi deficiency and blood stasis syndrome in the myocardial ischemia caused by coronary artery disease.2. Myocardial proteins results reflected the progress of the disease and myocardial ischemia syndrome evolution. The dynamic changes of cardiac proteins related to the syndromes dynamic evolution in myocardial ischemic disease.更多还原