新型心肌缺血模型建立及谷氨酰胺对糖尿病缺血复灌心肌的保护作用

发布时间：2018-01-06 04:23

本文关键词：新型心肌缺血模型建立及谷氨酰胺对糖尿病缺血复灌心肌的保护作用　出处：《北京协和医学院》2015年博士论文　论文类型：学位论文

【摘要】：人类疾病的动物模型是生物医学科学研究中所建立的具有人类疾病模似性表现的动物实验对象。现代生物医学研究中的动物模型的使用是一种最重要的实验方法,有助于更方便和有效地认识人类疾病,预防和控制疾病的发生和发展。随着中国老龄化的加剧、疾病谱的变化、饮食结构的改变,缺血心脏病发病率和死亡率正逐年增高。对缺血性心脏病的发病、诊断和治疗研究已经成为医学界热点,但是缺乏稳定可靠的标准化心肌缺血疾病模型是目前该领域研究的瓶颈。鉴于啮齿类小动物心脏结构和代谢与人类差距较大,小型猪心脏生理结构与病理变化与人类更为相似。因此我们利用微创技术(胸腔镜、小切口)和杂交手术技术,并对已有的建模工具进行改良和创新,共从三个角度建立了小型猪的心肌缺血模型：(1)小型猪缺血性二尖瓣返流模型(ischemic mitral regurgitation, IMR):经胸部小切口,利用了拥有自主知识产权的“房室瓣膜损伤器”进行二尖瓣后瓣拉伤,同时在冠脉回旋支起始部放置进口Ameroid环,模拟临床慢性心肌缺血后二尖瓣返流。(2)小型猪慢性心肌缺血模型(Chronic myocardial ischemia,CMI):针对进口Ameroid环的缺点,本研究对其结构进行改良,发明了新型的Ameroid环,并在胸腔镜下安放在冠脉回旋支,模拟临床慢性心肌缺血疾病过程。(3)糖尿病小型猪(急性)心肌缺血再灌注损伤(Acute ischemic reperfusioninjury, IRI)模型：首先静脉注射链脲佐菌素诱导糖尿病模型,建模成功后胸腔镜下微创冠脉阻断/开放前降支方法建立糖尿病小型猪缺血再灌注性损伤模型。模拟临床糖尿病下缺血再灌注损伤过程。针对各自模型的特点,我们对三种模型进行了详细的检查和评估(如血清学、超声、冠脉造影、核磁、PET/SPECT、病理学检查等)。结果发现利用微创技术和杂交技术建立的这三种心肌缺血模型均能够较好模拟人类疾病特点,具有相似性好、重复性好、可控性佳、手术简单易于操作等的特点,可以在基础实验和临床前实验中推广使用。糖尿病(Diabetes Mellitus, DM)是一种以胰岛素分泌和利用障碍、慢性高血糖为特征的代谢性疾病。糖尿病现在被认为是心血管疾病的主要的风险因素之一。前部分“糖尿病小型猪心肌缺血再灌注模型”实验也表明,糖尿病组术后梗死面积明显大于对照组,且并发症率也明显高于对照组。临床研究同样表明,合并糖尿病的冠心病患者血管再通后心血管事件的发生率及死亡率明显高于非糖尿病患者,这与糖尿病心肌缺血再灌注耐受性差密切相关,所以如何减轻糖尿病患者缺血再灌注损伤、减少心肌细胞凋亡是一个非常重要临床问题。对于糖尿病患者心肌缺血耐受性较差的这一特点,寻找潜在心肌缺血标志物和可能的治疗靶点,可能对此类患者的预后将会大有裨益。因此本研究基于小分子代谢产物可及时反映心肌组织对糖尿病和缺血再灌注损伤状态科学事实,首先利用前部分“糖尿病小型猪心肌缺血复灌模型”中灌装静脉窦血清和心肌标本,采用代谢组学研究方法,筛选糖尿病合并心肌缺血再灌注损伤病程中小分子差异性代谢产物。结果发现心肌和冠状静脉窦血液中分别存在12种和26中差异代谢产物,主要与糖、蛋白质、脂肪代谢、抗氧化应激等通路有关,提示这些代谢产物均在糖尿病心肌缺血再灌注中具有重要的作用。在发现的差异性代谢产物中,谷氨酰胺(Gin)在心肌中和冠状静脉窦血液中降低均极为明显(分别降低分别下降59.4%和73.4%),提示Gln在疾病的进程中发挥了关键作用。近期文献也表明Gin具有对缺血再灌注细胞的保护作用。但是在糖尿病病理情况下G1n是否也具有保护作用和具体机制,均未见报道。随后我们利用H9C2心肌细胞系和STZ诱导的糖尿病大鼠,分别在体内和体外两层面,研究G1n对糖尿病缺血再灌注心肌的作用。结果我们发现G1n能够升高H9C2细胞线粒体和胞浆中GSH浓度和GSH/GSSG比值,降低细胞和线粒体氧化应激,进而保护线粒体,减少细胞凋亡。体内实验中我们发现Gin能够减轻糖尿病大鼠缺血再灌注后机体氧化应激水平,改善心肌超微结构,减少心肌梗死面积,提高心脏术后收缩和舒张功能。所以Gln有希望成为临床上糖尿病患者减轻缺血再灌注风险的心肌保护性药物。
[Abstract]:The animal model of human disease is established by scientific research in animal experiments with object of human disease simulation performance. The use of animal models in modern biomedical research is one of the most important experimental method, contribute to a more convenient and effective understanding of human disease, occurrence and development of disease prevention and control. With China aging, changes in disease spectrum, the change of diet structure, ischemic heart disease morbidity and mortality are increasing year by year. The incidence of ischemic heart disease, diagnosis and treatment of the medical profession has become a hot spot, but the lack of a stable and reliable model of the standardization of myocardial ischemia disease is the bottleneck in the field. In view of the small rodent animal and human cardiac structure and metabolic gap, pig structure and pathology and physiological change of human heart is more similar. So we use micro A technology (thoracoscopic, small incision surgery) and hybrid technology, and modeling tools of improvement and innovation, miniature pig model of myocardial ischemia was established from three angles: (1) ischemic mitral regurgitation (ischemic mini pig model mitral regurgitation, IMR): the use of small incision in the chest, with independent intellectual property rights "atrioventricular valve injury" after mitral valve injury, while imports Ameroid ring placed in the circumflex coronary artery origin, clinical simulation of chronic myocardial ischemia after mitral regurgitation. (2) chronic myocardial ischemia model in pigs (Chronic myocardial ischemia, CMI): the imported Ameroid ring faults the research for the improvement of the structure, the invention of the Ameroid ring, and placed in the circumflex coronary artery in vats, simulate the process of clinical chronic myocardial ischemia disease. (3) Diabetic Minipigs (acute myocardial ischemia) Reperfusion injury (Acute ischemic, reperfusioninjury, IRI) model: the first intravenous injection of streptozotocin induced diabetic model, after the success of modeling thoracoscopic minimally invasive coronary artery occlusion / open anterior descending method to establish diabetic porcine ischemia reperfusion injury model. Simulated clinical diabetic ischemia reperfusion injury process. According to the characteristics of each model and we were examined and evaluated in detail on the three models (such as serology, ultrasound, coronary angiography, MRI, PET/SPECT, pathological examination). The results show that the three kinds of myocardial ischemia model was established using a minimally invasive technique and hybrid technology can simulate the characteristics of human disease, with similar good repeatability good, good controllability, operation is simple and easy to operate, can be widely used in experimental and pre clinical experiments. Diabetes (Diabetes Mellitus DM) is a kind of The use of insulin secretion and disorder of chronic hyperglycemia, metabolic disease characterized by diabetes. Now is considered to be one of the major risk factors for cardiovascular disease. The front part of "diabetic porcine model of myocardial ischemia reperfusion injury in experimental diabetic group also showed that postoperative infarct volume was significantly higher than the control group, and the complication rate is significantly higher than the control group. Clinical studies also show that the incidence and mortality of cardiovascular events in patients with coronary heart disease complicated with diabetic vascular recanalization after significantly higher than non-diabetic patients, the diabetic and myocardial ischemia reperfusion tolerance of difference are closely related, so how to reduce the ischemia reperfusion injury in diabetic patients, reduce myocardial apoptosis is a very important clinical for the characteristics of diabetic myocardial ischemia in patients with poor tolerance, looking for potential biomarkers of myocardial ischemia and possible treatment The target may, for such patients will be of great advantage. Therefore the prognosis based on small molecule metabolites can reflect the state of scientific fact of diabetes and myocardial tissue ischemia reperfusion injury, the former part of the myocardial ischemia reperfusion model in Diabetic Minipigs in filling venous sinus serum and myocardial specimens by metabonomics methods of screening of diabetes mellitus complicated with myocardial ischemia reperfusion injury and metabolic course of small molecular difference products. The results showed that the metabolites of 12 and 26 respectively in the myocardium and coronary venous sinus blood, mainly with sugar, protein, fat metabolism, anti oxidative stress pathway, suggesting that these metabolites play an important role in diabetes myocardial ischemia / reperfusion. Found in differences in metabolic products, glutamine (Gin) decreased in myocardial and coronary sinus in blood Are extremely obvious (respectively reduced down 59.4% and 73.4% respectively), suggesting that Gln plays a key role in the progression of the disease. The recent literature also suggests that Gin has a protective effect on ischemia reperfusion injury of cells. But in diabetic pathological cases, whether G1n has a protective effect and the specific mechanism, then we use are reported. Diabetic rat myocardial cell lines induced by STZ and H9C2, respectively, in vitro and in vivo in the two level, the effect of G1n on myocardial ischemia reperfusion in diabetic. Results we found that G1n can increase the H9C2 cell cytosol and GSH concentration and GSH/GSSG ratio, and decreased the cell mitochondrial oxidative stress, and protect the mitochondria, reduce cell in vivo apoptosis. We found that Gin can reduce ischemia reperfusion in diabetic rats after oxidative stress, improving myocardial ultrastructure, reduce myocardial infarct volume, extraction High cardiac contractile and diastolic function after high cardiac surgery. So Gln is expected to be a cardioprotective drug to reduce the risk of ischemia-reperfusion in patients with diabetes.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.2;R542.2;R-332

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