气虚血瘀证（心肌缺血）动物模型及蛋白质组学研究

【作者】 刘涛；

【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】 目的:证候理论是中医的核心理论之一,证候动物模型是揭示证候生物学基础的有力工具。我们的研究思路是以病理模型为研究载体,动态收集模型的“四诊”信息,建立以临床疾病和证候诊断标准为基础的评价体系,利用该体系对模型证候属性进行评价,同时应用蛋白质组学技术,从蛋白质差异表达的角度揭示证候的生物学基础。基于上述认识,本实验采用大鼠左冠状动脉前降支结扎模型,并从宏观表现、舌象、心功能、实验室指标和应证复方的疗效等方面对该模型进行证候属性的评价,认为该模型属于气虚血瘀证(心肌缺血),同时应用蛋白质组学技术,对假手术组和模型组心肌进行差异蛋白质群的检测,从蛋白质差异表达的角度揭示气虚血瘀证(心肌缺血)的生物学基础。方法:1造模方法和证候评价:采用雄性SD大鼠左冠状动脉前降支结扎的方法造模,以术后第3d出现4-6个异常Q波为冠脉结扎成功的标志,将成功的模型动物随机分为模型组、益气活血组和温阳利水组,同时制作假手术组(未结扎左冠状动脉前降支,其余与模型组相同),每组样本数为10,术后3天开始给药,灌胃体积为10ml/kg,假手术组和模型组10ml/kg饮用水灌胃,益气活血组给予芪参益气滴丸(天津天士力公司惠赠,主要成分黄芪、丹参、三七、降香)0.1g/kg灌胃,温阳利水组给予真武汤0.396g/kg灌胃,均相当于临床用量的6倍。给药周期为30天。证候属性的评价体系主要包括实验动物的宏观表现、舌象、心功能、实验室指标和应证复方的疗效等方面。术后第7、14、21、28d,观察实验动物的一般状态、舌象、爪甲和耳廓(将大鼠浅麻醉,标准色卡与鼠舌对比,用数码相机进行拍照,用Photoshop软件对舌的色度参数进行计算),检测体重、体温、呼吸频率、心电图(大鼠浅麻醉进行心电图收集)。术后8、15、22、29d,检测实验动物的游泳力竭时间(大鼠负重6%重物在温度30±1℃水中游泳,计算大鼠力竭时间,以大鼠头沉入水中10秒钟为力竭标志)。术后第30d,对实验动物进行超声心动图检测。术后第32d,实验动物处死,取大鼠舌尖组织作HE染色,并用放免的方法检测大鼠血浆ET、CGRP的含量。术后第32d,实验动物处死,取心肌组织分别作HE、Masson染色和TGF-β1免疫组化染色,并用放免的方法检测大鼠心肌组织TNF-α、IL-6、AngⅡ、Ald的含量。2气虚血瘀证(心肌缺血)蛋白质组学检测:术后第32天,模型组和假手术组各随机选择3只大鼠,模型组取大鼠心脏左室缺血部分,假手术组取左室正常区,分别作蛋白质双向电泳、银染、胶图分析和差异蛋白质的质谱检测。结果:1造模方法和证候评价:术后8、15、22、29d,模型组与假手术组相比游泳力竭时间显著缩短;术后22、29d,益气活血组和温阳利水组,与模型组相比均显著延长,二者之间无显著性差异。术后7、14、21、28天,模型组舌象与假手术组相比紫暗,部分出现瘀点、瘀斑, R、G、B和L显著下降;术后21、28d,益气活血组L值与模型组相比显著升高。与假手术组相比,模型组舌尖血管内径扩大,益气活血组与模型组相比,血管内径显著缩小。模型组与假手术组相比,血浆ET升高、CGRP下降;益气活血组与模型组相比,血浆ET显著下降、CGRP升高。术后第30d,模型组与假手术组相比,EF和FS显著下降;益气活血组与模型组相比,射血分数(EF)和短轴缩窄率(FS)显著增加。模型组与假手术组相比,TNF-α、IL-6、AngⅡ、Ald显著增高;益气活血组与模型组相比,TNF-α、IL-6、AngⅡ、Ald均明显下降。模型组与假手术组相比,CVF(胶原容积百分比)显著增加;益气活血组CVF与模型组相比显著减少。TGF-β1免疫组化结果显示,与假手术相比,模型组心肌组织TGF-β1表达明显增多(P<0.05);益气活血组心肌组织TGF-β1表达与模型组相比显著下降。以上结果说明,结扎大鼠左冠状动脉前降支模型属于气虚血瘀证模型。2气虚血瘀证(心肌缺血)蛋白质组学检测:气虚血瘀证(心肌缺血)模型组缺血区心肌组织苹果酸脱氢酶、铁硫蛋白8、肌浆球蛋白轻链、谷胱甘肽过氧化物酶、铜锌超氧化物歧化酶含量下降,αB-晶状体蛋白、磷酸丙糖异构酶、蛋白酶前体或α亚基含量增高。气虚血瘀证(心肌缺血)缺血区心肌蛋白质表达与能量代谢、氧化损伤以及心肌自我保护蛋白质有关。结论:1通过对左冠状动脉前降支大鼠模型从宏观表现、舌象、心功能、实验室指标和应证复方的疗效进行证候的动态观察和评价,结果显示该模型属于气虚血瘀证(心肌缺血)。2气虚血瘀证(心肌缺血)标志蛋白质群与能量代谢、氧化损伤以及心肌自我保护蛋白质有关。气虚血瘀证(心肌缺血)的外在表现是以其心肌细胞能量代谢障碍、氧化损伤、心肌细胞坏死以及心肌间质纤维化等改变为基础的。更多还原

【Abstract】 Object : Syndromes theory is the core theory of Chinese medicine theory . Syndromes animal model is a powerful tool for revealing syndrome biological basis . The overall and dynamic characteristics of Proteomics is surprisingly similar with the "whole concept" and the "concept of differentiation"of Chinese medicine theoretical system. Based on the above understanding, the rat model of left anterior descending coronary artery ligation is applied and judged by Syndrome property assessment from macro-performance, tongue, cardiac function、laboratory indicators and the efficacy of traditional Chinese composite prescriotion .The model of myocardial ischemia was sured to belongs to Qi-deficiency and Blood–stasis Syndrome . We discuss the pharmacological mechanism of replenishing Qi and activating Blood composite prescriotion to reverse myocardial fibrosis . And we applied proteomics technology to detect the cardiac different protein group between sham-operated group and the model group to reveal the biological basis of Qi-deficiency and Blood–stasis Syndrome.Methods :1 Methods of constrcting model and collecting four diagnostic information: left anterior descending coronary artery ligation rat model was applied.3 days after surgery , 4-6 abnormal Q–wave showed that coronary ligation is successful,Successful models were randomly divided into three groups, model group、Replenishing Qi and Activating Blood (RQAB)group and Warming yang to excrete water(WYEW) group,while producing a sham -operated group (not ligation of the left anterior descending coronary artery, and the rest is the same with model group).3 days after surgery, sham-operated group and the model group were stomach-perfused for 10ml/kg.d drinking water , RQAB group for 0.1g/kg.Qishenyiqi Pills (the main component of astragalus root, red sage root, notoginseng. dalbergia wood) WYEW group for 0.396g/kg.d Zhen Wu decoction. The experimental cycle is 30 days. At the end of the experiment, the number of each group sample is 10.At the 7、14、21、28 day of postsurgery,we observed and detected the rats of the general state, the tongue, fingernail and auricular, body weight, body temperature, respiratory rate and ECG. At the 8、15、22、29 day of postsurgery,we detected the rats, exhaustive swimming time (6% in heavy load ,temperature of 30±1°C water swimming, Exhaustive in terms of time, sinking into the water in the first 10 seconds as the exhaustive identification).At the 30 day of postsurgery ,the rats echocardiography was detected. At the 32 day of postsurgery ,the rats were killed, and the tongue tissue was dealed with HE staining, and the ET and CGRP of plasma was detected by RIA.Myocardial tissue were dealed with HE and Masson staining . The expression of TGF-β1 was detected by immunohistochemical staining, and the TNF, IL-6, Ang II, Ald of rat myocardial tissue were detected by RIA.2 Methods of detecting myocardial Proteomics : At the 32 day of postsurgery, three rats were randomly selected in the model group and the sham-operated group.The rat of model group chose the myocardial tissue of ischemic area ,and the rat of sham-operated group normal left ventricular The above myocardial tissues were dealed with the two-phase protein electrophoresis、silver staining and protein mass spectrometry testing.Result:1 the evaluation of syndrome : At the 8、15、22、29 day of postsurgery, compared with the sham-operation group ,model group ,s exhaustive swimming time decreased significantly; At the 22、29 day of postsurgery, RQAB group and WYEW group,s exhaustive swimming time increased significantly compared with model group. The model group ,s tongues were dark and purple compared with the sham-operation group, and R, G, B and L value of the model group ,s tongues dropped significantly ; At the 21,28d of postsurgery, L value of RQAB group increased significantly compared with the model group . Compared with the sham-operated group, the micro-vein diameter of the model group increased significantly ; Compared with the model group ,the micro-vein diameter of RQAB group decreased significantly (P <0.05). Compared with the sham-operated group, ET of the m odel group increased and CGRP decreased; Compared with the model group ,ET and CGRP increased . Compared with the sham-operated group ,EF and FS of the m odel group decreased ; Compared with the model group , EF and FS of the RQAB group increased the model group and the sham-operation group, dropped significantly.Compared with the sham-operated group, TNF, IL-6, Ang II, Ald of model group increased significantly; Compared with the model group , TNF, IL-6, Ang II, Ald of the RQAB group decreased . Compared with the sham-operated group, CVF of model group increased significantly; Compared with the model group , CVF of the RQAB group decreased . Compared with the sham-operated group, the expression of TGF-β1 of model group increased significantly; Compared with the model group , the expression of TGF-β1 of the RQAB group decreased .2 proteomics results of Qi-deficiency and Blood–stasis syndrome : Compared with the model group,the ischemic myocardium , s MDH, 8 iron-sulfur protein, myosin light chain, glutathione peroxidase, Cu-Zn superoxide dismutase decreased in the model group,αB-crystallin protein,triosephosphate isomerase,protease subunit precursor increased .Conclusion:1 The model of myocardial ischemia (left anterior descending coronary artery ligation rat model )belongs to Qi-deficiency and Blood–stasis Syndrome2 The diffenret protein group of myocardial ischemia Qi-deficiency and Blood–stasis Syndrome are related with the proteins of energy metabolism, oxidative damage and myocardial self protection.The external manifestation of myocardial ischemia Qi-deficiency and Blood–stasis Syndrome is based on the changes of myocardial energy metabolism, oxidative damage, myocardial necrosis and myocardial fibrosis.更多还原