本文關(guān)鍵詞：新型心肌缺血模型建立及谷氨酰胺對糖尿病缺血復灌心肌的保護作用　出處：《北京協(xié)和醫(yī)學院》2015年博士論文　論文類型：學位論文

  更多相關(guān)文章： 動物疾病模型 心肌缺血 小型豬 微創(chuàng)手術(shù) 代謝組學 谷氨酰胺 線粒體 氧化應激

【摘要】：人類疾病的動物模型是生物醫(yī)學科學研究中所建立的具有人類疾病模似性表現(xiàn)的動物實驗對象。現(xiàn)代生物醫(yī)學研究中的動物模型的使用是一種最重要的實驗方法,有助于更方便和有效地認識人類疾病,預防和控制疾病的發(fā)生和發(fā)展。隨著中國老齡化的加劇、疾病譜的變化、飲食結(jié)構(gòu)的改變,缺血心臟病發(fā)病率和死亡率正逐年增高。對缺血性心臟病的發(fā)病、診斷和治療研究已經(jīng)成為醫(yī)學界熱點,但是缺乏穩(wěn)定可靠的標準化心肌缺血疾病模型是目前該領(lǐng)域研究的瓶頸。鑒于嚙齒類小動物心臟結(jié)構(gòu)和代謝與人類差距較大,小型豬心臟生理結(jié)構(gòu)與病理變化與人類更為相似。因此我們利用微創(chuàng)技術(shù)(胸腔鏡、小切口)和雜交手術(shù)技術(shù),并對已有的建模工具進行改良和創(chuàng)新,共從三個角度建立了小型豬的心肌缺血模型：(1)小型豬缺血性二尖瓣返流模型(ischemic mitral regurgitation, IMR):經(jīng)胸部小切口,利用了擁有自主知識產(chǎn)權(quán)的“房室瓣膜損傷器”進行二尖瓣后瓣拉傷,同時在冠脈回旋支起始部放置進口Ameroid環(huán),模擬臨床慢性心肌缺血后二尖瓣返流。(2)小型豬慢性心肌缺血模型(Chronic myocardial ischemia,CMI):針對進口Ameroid環(huán)的缺點,本研究對其結(jié)構(gòu)進行改良,發(fā)明了新型的Ameroid環(huán),并在胸腔鏡下安放在冠脈回旋支,模擬臨床慢性心肌缺血疾病過程。(3)糖尿病小型豬(急性)心肌缺血再灌注損傷(Acute ischemic reperfusioninjury, IRI)模型：首先靜脈注射鏈脲佐菌素誘導糖尿病模型,建模成功后胸腔鏡下微創(chuàng)冠脈阻斷/開放前降支方法建立糖尿病小型豬缺血再灌注性損傷模型。模擬臨床糖尿病下缺血再灌注損傷過程。針對各自模型的特點,我們對三種模型進行了詳細的檢查和評估(如血清學、超聲、冠脈造影、核磁、PET/SPECT、病理學檢查等)。結(jié)果發(fā)現(xiàn)利用微創(chuàng)技術(shù)和雜交技術(shù)建立的這三種心肌缺血模型均能夠較好模擬人類疾病特點,具有相似性好、重復性好、可控性佳、手術(shù)簡單易于操作等的特點,可以在基礎(chǔ)實驗和臨床前實驗中推廣使用。糖尿病(Diabetes Mellitus, DM)是一種以胰島素分泌和利用障礙、慢性高血糖為特征的代謝性疾病。糖尿病現(xiàn)在被認為是心血管疾病的主要的風險因素之一。前部分“糖尿病小型豬心肌缺血再灌注模型”實驗也表明,糖尿病組術(shù)后梗死面積明顯大于對照組,且并發(fā)癥率也明顯高于對照組。臨床研究同樣表明,合并糖尿病的冠心病患者血管再通后心血管事件的發(fā)生率及死亡率明顯高于非糖尿病患者,這與糖尿病心肌缺血再灌注耐受性差密切相關(guān),所以如何減輕糖尿病患者缺血再灌注損傷、減少心肌細胞凋亡是一個非常重要臨床問題。對于糖尿病患者心肌缺血耐受性較差的這一特點,尋找潛在心肌缺血標志物和可能的治療靶點,可能對此類患者的預后將會大有裨益。因此本研究基于小分子代謝產(chǎn)物可及時反映心肌組織對糖尿病和缺血再灌注損傷狀態(tài)科學事實,首先利用前部分“糖尿病小型豬心肌缺血復灌模型”中灌裝靜脈竇血清和心肌標本,采用代謝組學研究方法,篩選糖尿病合并心肌缺血再灌注損傷病程中小分子差異性代謝產(chǎn)物。結(jié)果發(fā)現(xiàn)心肌和冠狀靜脈竇血液中分別存在12種和26中差異代謝產(chǎn)物,主要與糖、蛋白質(zhì)、脂肪代謝、抗氧化應激等通路有關(guān),提示這些代謝產(chǎn)物均在糖尿病心肌缺血再灌注中具有重要的作用。在發(fā)現(xiàn)的差異性代謝產(chǎn)物中,谷氨酰胺(Gin)在心肌中和冠狀靜脈竇血液中降低均極為明顯(分別降低分別下降59.4%和73.4%),提示Gln在疾病的進程中發(fā)揮了關(guān)鍵作用。近期文獻也表明Gin具有對缺血再灌注細胞的保護作用。但是在糖尿病病理情況下G1n是否也具有保護作用和具體機制,均未見報道。隨后我們利用H9C2心肌細胞系和STZ誘導的糖尿病大鼠,分別在體內(nèi)和體外兩層面,研究G1n對糖尿病缺血再灌注心肌的作用。結(jié)果我們發(fā)現(xiàn)G1n能夠升高H9C2細胞線粒體和胞漿中GSH濃度和GSH/GSSG比值,降低細胞和線粒體氧化應激,進而保護線粒體,減少細胞凋亡。體內(nèi)實驗中我們發(fā)現(xiàn)Gin能夠減輕糖尿病大鼠缺血再灌注后機體氧化應激水平,改善心肌超微結(jié)構(gòu),減少心肌梗死面積,提高心臟術(shù)后收縮和舒張功能。所以Gln有希望成為臨床上糖尿病患者減輕缺血再灌注風險的心肌保護性藥物。
[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.

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R587.2;R542.2;R-332

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