【作者】 郭淑贞；

【导师】 王伟；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2007， 博士

【摘要】 证候是中医药理论的核心内容,证候科学内涵的揭示是中医基础理论现代化的关键环节,而证候生物学基础研究的突破则是证候科学内涵得以阐明的保障。证候生物学基础的研究同样需要借助动物模型这个必须的工具。目前,证候动物模型的建立和评价存在巨大的争议,缺乏公认的标准,亟待寻找可行的途径。通过在疾病模型的基础上,对模型动物进行动态的观察、评价以确定其证候属性的方法,有可能建立符合中医理论且稳定性较好的病证结合动物模型。同时,以往证候的生物学基础研究多致力于寻找可用于证候诊断的金指标,但由于忽略了证候的整体性而遇到了瓶颈。系统生物学研究技术和方法的兴起和发展,为证候生物学基础的研究带来了新的契机。循环系统作为机体各种组织、器官、细胞联系的纽带,在机体的生命活动过程中起着重要的作用,更能反映机体整体的生理病理状态。因此,对于证候相关的血浆蛋白质组学的研究有可能发现证候在蛋白质层面的特征模式。血瘀证的是冠心病的最常见的证型,同时有着较好的研究基础,因此血瘀证的研究可作为证候动物模型研究的切入点,即通过对心肌缺血模型动态观察确定血瘀证时间窗的方式来建立血瘀证(心肌缺血)动物模型,并对血瘀证(心肌缺血)动物血浆蛋白质组学进行研究,以探讨将蛋白质组学技术应用于证候生物学基础研究的可行性。目的:建立稳定的血瘀证(心肌缺血)动物模型,并寻找其相关的血浆蛋白质组学特征。方法:1小型猪血瘀证(心肌缺血)模型的建立和评价中国农业大学育种健康中华小型猪,体重25±4kg,月龄6-10个月,雌雄不限,随机分为模型组和假手术组。模型组动物在麻醉、无菌条件下开胸,分离前降支,在第一对角支远端的前降支主干放置Ameroid缩窄环,关胸。假手术组除不放置缩窄环外,其余均同模型组。1.1参考前期动态观察的实验结果,在术后4周采集动物四诊信息,进行心电图、超声心动、冠脉造影等检查,并在前腔静脉采血,检测血液流变学指标以及前列环素(PGI2)、血栓素A2(TXA2)、内皮素(ET-1)、降钙素基因相关肽(CGRP)等血管活性物质的含量。参考上述指征,对模型动物的疾病类型及证候属性进行诊断。1.2诊断为心肌缺血血瘀证的模型动物随机分为活血化瘀组及模型组,每组6只。活血化瘀组动物每日喂饲丹七片9g/只,用药4周,饮食及饲养条件同模型组及假手术。实验终点采集动物四诊信息,进行心电图、超声心动、冠脉造影等检查,并在前腔静脉采血,检测血液流变学指标以及PGI2、TXA2、ET-1、CGRP等血管活性物质的含量,根据上述信息再次对模型动物的疾病及证候进行诊断,以确定模型动物表现为心肌缺血血瘀证的时间窗。在实验终点对心肌组织采用常规石蜡包埋,HE、Masson染色观察其形态学变化。观察丹七片对对上述指标的影响,评价其对心肌缺血以及血瘀证的改善作用,以增加证候诊断的证据。2小型猪血瘀证(心肌缺血)的血浆差异蛋白质组学研究术后4周,前腔静脉采血,枸橼酸钠抗凝,离心收集上清备用。血浆总蛋白采用Bradford法测定,加样1.3mg。PH范围3-11NL 24cmIPG胶条等电聚焦,1mm、13% SDS-PAGE二相分离。考马斯亮蓝R350染色,凝胶扫描后,采用Imagemaster 2D Platinum 6.0软件进行凝胶的蛋白点检测、匹配。对匹配蛋白点的体积百分比进行比较,确定差异蛋白点。差异蛋白点胰蛋白酶酶切后,MALDI-TOF检测,数据库搜索,确定其相关信息。术后8周,分别采集模型组、假手术组、活血化瘀组动物血浆样本,并进行双向凝胶电泳分析及蛋白点的检测、匹配,方法同前。通过与手术4周时凝胶的匹配,观察术后4周存在的差异蛋白点在8周模型中的变化以及活血化瘀药物对这些蛋白的影响。结果:1小型猪血瘀证(心肌缺血)模型的建立和评价1.1术后4周模型评价1.1.1四诊信息:模型动物出现精神紧张、易激惹,怕人,不易接近,冲撞笼栏;被毛杂乱、无光泽等表现,舌色略见暗红或青紫;假手术动物精神逐渐恢复正常,进食、饮水量增加,被毛整齐,有光泽,舌色淡红或淡白。1.1.2心电图:模型组动物多在V3、V4导联出现ST段水平或斜行向下压低达0.1mV,同时出现多导联的T波倒置;部分动物出现异常Q波。在观察期间模型动物未发现明显的心律失常。1.1.3超声心动图:与假手术组相比,模型动物心脏超声的特征主要表现为左室腔结构的变化,即左室舒张末、收缩末内径( P < 0.01)及容积( P <0.05 )的扩大。左室前壁乳头肌及心尖平面收缩末及舒张末厚度的减小,以收缩末的变化更为显著( P <0.01 )。同时,心尖水平的左室前壁收缩期室壁增厚率减小( P <0.01 )。另外,室间隔收缩末厚度减小( P <0.05 )。心脏整体的功能包括SV、EF、FS以及E/A比等均无明显变化,提示其心功能仍处于代偿期。1.1.4冠脉造影:模型动物前降支狭窄率在90%以上,甚至完全闭塞,TIMI血流分级多为Ⅰ级和Ⅱ级。假手术组前降支充盈良好,TIMI血流Ⅲ级。1.1.5血液流变学:与假手术组相比,模型动物的血液流变学特征主要表现为高(P <0.05)、中(P <0.01)、低(P <0.01)切变率下全血黏度的升高,而其红细胞的聚集以及变形指数等无明显差异。1.1.6其它血液理化指标:与假手术组相比,模型组动物的血浆6-K-PGF1α显著降低(P<0.05),TXB2、K/T无显著差异;血浆CGRP、ET、E/C均无明显变化。1.2术后8周模型评价1.2.1术后第8周,模型动物精神状况有所恢复,饮食、二便接近正常;被毛杂乱、无光泽等表现仍然存在,舌象仍表现为舌色略见暗红或青紫,无明显的瘀点瘀斑。1.2.2心电图:模型组动物心电图与术后4周相近。1.2.3超声心动图:模型组与假手术组相比,与术后4周存在相似的差异。1.2.4血液流变学:模型组动物高、中、低切变率下的全血黏度较假手术组显著增高(P<0.01),红细胞压积较假手术组显著升高(P<0.05)。1.2.5其它血液理化指标:术后8周,模型组动物血浆6-K-PGF1α水平较假手术组显著降低(P<0.05)。1.3活血化瘀药物丹七片的治疗作用1.3.1活血化瘀组动物四诊信息、心电图与模型组相比无显著差异。1.3.2超声心动图:模型组室间隔收缩末厚度较假手术组显著减小( P <0.05 ),而活血化瘀组与假手术组相比无显著差异。1.3.3血液流变学:与模型组相比,活血化瘀组红细胞压积测定值显著回落(P<0.05),且与假手术组相比无显著差异;活血化瘀组红细胞变形指数较模型组显著增加(P<0.05)。1.3.4其它理化指标:与模型组相比,活血化瘀组动物血浆6-K-PGF1α显著回升(P<0.05),与假手术组无显著差异。1.3.5病理学观察:HE染色见假手术组动物心肌排列整齐、结构清晰;模型动物病变区表现为心肌细胞排列紊乱,心肌细胞肿胀、变性、坏死,炎细胞侵润;活血化瘀组动物有明显的改善。Masson染色见模型组动物病变区心肌间质胶原组织增多,心肌细胞结构紊乱,细胞核或细胞浆空泡变性,破坏的心肌被胶原组织取代,同时可见炎细胞浸润;活血化瘀组动物心肌胶原沉积区数量和面积均小于模型组。2小型猪心肌缺血血瘀证的血浆差异蛋白质组学研究在术后4周混合样品的电泳发现模型组有10个明显的上调蛋白点;在单一血浆样品的电泳发现9个明显的上调蛋白点。其中1个蛋白点在两次实验中均有差异。质谱鉴定结果提示,部分上调的差异蛋白为α1-酸性糖蛋白、α1-抗胰蛋白酶、胎球蛋白A、载脂蛋白A-I、载脂蛋白A-IV、补体C4、补体细胞溶解抑制因子。模型组在术后4周存在的部分差异蛋白点在术后8周仍有差异,但其相对含量与术后4周不同。而活血化瘀药物只对上述部分差异蛋白有影响。结论:1模型动物在术后4周,可诊断为心肌缺血血瘀证,且该诊断在术后8周仍成立。提示在该时间窗,此类动物模型稳定表现为血瘀证(心肌缺血)。丹七片可以改善心肌缺血及血瘀证的部分表现,在一定程度上佐证了上述诊断。2模型组与假手术组相比,已鉴定的差异蛋白质主要为急性期反应蛋白、补体系统以及载脂蛋白等,炎症/抗炎是其互相联系的核心环节,提示在该心肌缺血血瘀证同时存在着炎症反应这一病理进程,炎症反应可能是血瘀证的病理机制之一,但仍有待进一步研究。这些蛋白在术后8周则表现为这些蛋白不同的组合模式,但仍维持了其基本特征。提示证候的蛋白质组学特征可能是某些差异蛋白及其组合模式。丹七片只对部分血浆差异蛋白有影响,提示这些已鉴定可能的血瘀证差异蛋白并不全是其效应靶点。更多还原

【Abstract】 Since syndrome is the core theory of Chinese medicine, the revealing of its scientific connotation is considered as the key point to the modernization of Chinese medicine. However it depends on the breakthrough on the biology basis research of syndromes. As other biological researches, animal models are necessary. Considerd to the lack of recognized standards, there is a huge controversy in practical ways to establish or evaluate syndrome animal models. so, an objective and admitted standard is necessary. We will try to determine its syndromes’attributes based on dynamic observation and strict evaluation on a disease animal model, which may be a practical way to establish an integrated disease and sydrome animal model with more reproducibility and better accordance to the theories of Chinese medicine.Meanwhile, research on the biological basis of syndromes encountered a bottleneck due to the aims to find single indicator for the diagnosis of syndromes in the past years. However syndrome is an integritical concept and its scientific content may need an overall review of the organism. There is a beam of hope now attributing to development of systematic biology research techniques and methods. Plasma is one of the most informative bodily fluids, as it provides a link between human organs, tissues and cells. Because of its important role in vital movement, there is great information of the overall physiological and pathological conditions in plasma. Therefore, the plasma proteomic research may provide some evidence for the protein patterns of syndrome.Blood stasis is the most common syndrome type of coronary heart disease, and there were many related researches before. So the research on the animal model of blood stasis syndrome can be used as the starting point. The time window of blood stasis in a myocardial ischemia model was determined depend on the dynamic observation, and then the characteristics of its plasma proteome was explored.Aims:To establish a stable blood stasis （myocardial ischemia） animal model, and find the characteristics of its plasma proteome.Methods:1 Establishment and assessment of a blood stasis model with myocardial ischemiaChinese experimental mini swines （25±4 kg） were instrumented with a size-matched Ameroid constrictor on the anterior descending branch under general anesthesia in sterile conditions. And a sham group was also included. Based on early results of dynamic observations, evaluations were performed four weeks after operation. Clinical performances of animals were collected and electrocardiogram, echocardiography, coronary angiography were detected. Meanwhile, blood was obtained from former cava vena to detect blood rheological features and the concentration of prostacyclin, thromboxane A2, endothelin-1 and calcitonin gene-related peptide. Then the diseases of model animals were diagnosed and their syndromes were differentiated.Then, blood stasis animals with myocardial ischemia were randomly divided into therapeutic group and control group （n = 6）. Nine gram Danqi tablet were administrated once a day to a therapeutic animal for 4 weeks. At the end of experiment, to determine the diagnosis of control animals and the effect of Danqi tablet, all animals were evaluated in the same way as which performed 4 weeks after operation. After sacrificed, myocardial samples destined to histological evaluation were fixed in 4% phosphate buffered paraformaldehyde, routinely dehydrated and embedded in paraffin. Then serial sections were stained with HE stain and Masson’s trichrome stain to evaluate the general morphology.2 Research on plasma proteome of swines of blood stasis with myocardial ischemiaBlood was obtained from the animals with blood stasis syndrome in myocardial ischemia and matched sham operated animals 4 weeks after operation. Plasma samples were centrifuged and then stored at -80℃.All plasma samples were quantitated by Bradford assay, and then separated by two-dimensional electrophoresis （2-DE） to identify differentially expressed proteins. Precast IPG strips （pH3-11 nonlinear, 24 cm） were used in the first dimension, and SDS-polyacrylamide gel electrophoresis （PAGE） was performed using 1 mm thick, 13% SDS-PAGE gels. The Coomassie Brilliant Blue R350-stained 2-DE were scanned with digital scanner and analyzed with ImageMaster 2D Platinum 6.0 software. A cluster of protein spots differentially expressed were selected and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）.After 4 weeks’therapy of Danqi tablet, plasma samples were obtained from animals in therapeutic, control and sham operated group, and then analyzed by 2-DE as described above to evaluate the differentially expressed proteins got from previous experiment.Results:1 Establishment and assessment of a blood stasis model with myocardial ischemia1.1 Evaluations performed 4 weeks after operation1.1.1 Clinical performances:Model animals appeared mental stress, irritation, fear, violent behaviors, strong self-defense, pilose disorderly and lack of luster, slightly dark red or purple tongue, etc, while sham operated animals’performances gradually returned to normal, with increased appetite, neat and shiny pilose, light red or light white tongue.1.1.2 ECG:More than 0.1mV ST segment depression and inversion T waves were observed in several leads of surface electrocardiogram of model animals. But no significant arrhythmia was found in experimental period.1.1.3 EchocardiographyThe structure and function of left ventricular were evaluated by echocardiography. Compared with sham operated animals, there was great increase of end-systolic volume （P < 0.05）, end-diastolic volume （P < 0.05）, end-systolic diameter （P < 0.01） and end-diastolic diameter （P < 0.01） in model animals. Anterior wall thickness of both papillary muscle and apex level was decreased at end-diastolic and end-systolic in model animals （P < 0.01）. Meanwhile, the apex of the left ventricular anterior systolic wall thickening decreased （P < 0.01）. In addition, septal thickness at the end-systolic decreased （P < 0.05）. All the results above showed a segmental dysfunction of the left ventricular. There were no significant changes in stroke volume, ejection faction, fraction of shortening and E/A, which suggested that cardiac function was still in compensatory period.1.1.4 Coronary angiographyExvivo angiography performed more than 90% occlusion or even completely block of the left anterior descending artery by the Ameroid constrictor in model animals. TIMI flow gradeⅠorⅡwere observed in most model animals. There was good filling the in left anterior descending artery with TIMI flow gradeⅢin Sham-operated animals.1.1.5 HemorrheologyCompared with sham-operated animals, the blood viscosity of model animals increased significantly at a shear rate of high （P < 0.05）, mid （P < 0.01）, low （P < 0.01）. No other changes were observed.1.1.6 Other changesCompared with sham-operated animals, the concentration of 6-K-PGF_1α in plasma decreased significantly（P < 0.05）, while other factors observed did not have significant change.1.2 Evaluations performed 8 weeks after operationCompared with sham-operated group, the most changes of model group detected at 4 week still existed at 8 weeks except the clinic performances. Model animals seemed recovering a little, such as appetite, urine and stool. But the other characters were also persistent such as disorderly and lackluster clothing hair, slightly dark red or purple tongue, low dose of 6-K-PGF1α, changes of ECG, echocardiography and hemorrheology, etc.1.3 Effect of Danqi tabletDue to treatment of Danqi tablet, red blood cell deformation was improved（P < 0.05）, elevated volume of packed red blood cells was decreased（P < 0.05）, and the decreased 6-K-PGF1αwas recovered（P < 0.05）, when compared with model animals. Compared with sham oprated animals, the septal thickness of model animals at the end-systolic decreased （P < 0.05） while that of thearaputic animals didn’t.The main lesion of model animal heart observed in HE staining were disarranged, swell, degeneration, necrosis myocardial cells, and infiltrated inflammatory cells. These lesions were improved in slices of therapeutic animals. In the Masson trichrome-stained slices of ischemia region of model animals’heart, increased collagen in intercellular, infiltrated inflammatory cells, injured myocardial cells with vacuolar degeneration, disarranged structure, or displaced by collagen were observed. Meanwhile in the slices of therapeutic animals, the number and size of the collagen hosted region were less.2 Differential express of plasma proteome in model animals10 up-regulated protein spots were detected in the 2-DE separation of pooled samples, while 9 up-regulated in single plasma sample test. Among these differential expressed proteins, 1 was existed in both tests. MS results suggest that the unregulated proteins includingα1-acid glycoprotein,α1-antitrypsin, fetuin A, apolipoprotein A-IV, apolipoprotein A-I, Complement C4, complement cytolysis inhibitor. Other changes were still unknown.Part of the differential expressed proteins detected in 4weeks’animal models were still differential ones with different relative concentration in 8 weeks’models. However, significant improvement was observed only in part of these proteins of Danqi treated animals.Conclusions:1 The Chinese experimental miniature swine with Ameroid constrictor around the left descending artery were performed as blood stasis syndrome with myocardial ischemia between 4 and 8 weeks after operation. Danqi tablet improved some indexes observed, which provided other evidences of the diagnosis.2 The detected differential expressed plasma proteins of animals with blood stasis syndrome were associated with inflammatory reactions. Thus, we supposed that inflammation may be one of the important pathologic processes in blood stasis syndrome. However, their exact relationship was still unknown.The proteomic feature of syndrome may be some differential expressed proteins and their different assemblies.Danqi tablet only showd effect on part of differential expressed proteins, which suggested that some of these proteins were not effective targets of Danqi tablet.更多还原