谷氨酰胺抗高糖心肌缺血再灌注损伤机制及心肌缺血新模型研究

发布时间：2018-07-10 12:10

本文选题：转化生长因子-β1-Smad3通路 + 谷氨酰胺　；参考：《北京协和医学院》2016年博士论文

【摘要】：背景：转化生长因子-β1 (transforming growth factor-β1, TGF-β1)-Smad3通路的激活可以加重心肌缺血再灌注损伤。糖尿病患者的心肌组织中TGF-β1-Smad3通路被明显地激活,这极有可能成为糖尿病患者经历心肌缺血再灌注损伤预后较差的重要原因之一。我们前期的体外实验已经证明了谷氨酰胺可以减轻高糖联合缺氧复氧所导致的心肌细胞损伤。本次实验的研究目的是进一步深入研究谷氨酰胺能否可以通过抑制TGF-β1-Smad3这条通路,发挥抗糖尿病心肌缺血再灌注损伤的作用。方法：体外实验里,我们将H9c2大鼠心肌细胞置于33mM的高糖环境下培养,然后进行缺氧复氧和对照处理。首先,给予婧GF-β1受体抑制剂SB431542和Smad3的特异性抑制剂SIS3对Smad3通路进行抑制,观察细胞损伤变化。其次,我们使用不同浓度的谷氨酰胺进行干预治疗观察对心肌细胞Smad3通路的影响。最后,我们又应用人重组的TGF-β1激活Smad3通路来进行验证。体内实验中,我们首先建立糖尿病大鼠模型；然后对正常大鼠和糖尿病大鼠分别给予生理盐水或谷氨酰胺溶液灌胃处理；最后再进行心肌缺血再灌注损伤的干预。我们应用Western blotting技术检测了H9c2心肌细胞和大鼠心肌组织中TGF-β1,总Smad3,磷酸化Smad3和活化的caspase-3的表达水平。应用TUNEL法检测了心肌细胞和心肌组织中的细胞凋亡率。我们也检测了大鼠心肌梗死面积和电镜下心肌微观结构的变化以及大鼠的相关血流动力学指标参数的变化。结果：研究发现：在体外实验中,高糖+缺氧复氧组中的H9c2细胞的凋亡率最高,同样该组的TGF-β1-Smad3通路被激活的也最明显。TGF-β1受体抑制剂SB431542和Smad3的特异性抑制剂SIS3均可以明显下调Smad3的磷酸化水平,并能够有效减轻对心肌细胞的高糖+缺氧复氧损伤。同样地,补充谷氨酰胺后,细胞的凋亡水平下降了,并且TGF-β1-Smad3通路的激活也被抑制了。然而,当外源给予人重组TGF-β1后,谷氨酰胺的保护作用则削弱了,心肌细胞凋亡水平明显增加。在体内实验中,非谷氨酰胺治疗组的糖尿病大鼠经历心肌缺血再灌注后心脏损伤最严重。对糖尿病大鼠进行补充谷氨酰胺治疗可以明显改善心肌缺血再灌注后的血流动力学指标,减少心肌细胞凋亡、心肌微观结构损害与心肌梗死面积,同时也抑制了心肌组织中TGF-β1-Smad3通路的激活。结论：因此,我们得出以下结论：TGF-β1-Smad3通路的激活可以加重糖尿病心肌缺血再灌注损伤;补充谷氨酰胺可以部分通过抑制TGF-β1-Smad3通路减轻了糖尿病心肌缺血再灌注损伤。背景：在论文第一部分的研究中,我们采用了SD大鼠建立了心肌缺血模型。这种小型啮齿类动物的心脏大小及结构与人类心脏相差较大,在一些实验应用方面受到限制。而某些大型动物则体现出独特的优势。如比格犬,它具有与人类相似的心脏结构及病理生理反应,且循环系统发达,更适合于心血管疾病的研究。比格犬遗传性能稳定且优良,也为实验的均一性也提供了保障。目前缺少一种与临床状态相近的,可以评价溶栓疗效的心肌缺血动物模型。所以在论文第二部分实验中,我们首先分析了临床中冠状动脉血栓的成分后,然后利用微创介入技术在冠状动脉内注入自体栓子的方法研发了一种能够评价溶栓药物疗效的比格犬心肌缺血模型,也为后续探讨溶栓后的缺血再灌注损伤相关机制奠定了实验基础。方法：我们选取了18只比格犬进行了此次实验,一共分为3组：红色血栓组(n=6只),白色血栓组(n=6只)和白色血栓+rt-PA溶栓再灌注组(n=6只)。自制的自体血栓被注入到冠状动脉前降支的中远段。通过心电图和心电监护仪监测冠状动脉栓塞及溶栓再灌注过程。应用冠状动脉造影确定血栓在体状态。应用HE染色和电镜技术对不同血栓成分进行分析。应用2,3,5-氯化三苯基四氮唑(TTC)染色计算心肌梗死面积。结果:经过HE染色和扫描电镜分析发现,红色血栓具备松散的网状结构特点,网眼中充满红细胞；而白色血栓的结构较为紧密,它主要有密集的纤维蛋白构成。冠状动脉造影显示,当冠状动脉栓塞3小时后,红色血栓组的再通率(自溶率)为2/6,而白色血栓组再通率为0/6。并且红色血栓组再通的2例均出现了心律失常,心电图表现出升高的ST段回落和原理高耸的T波出现降低,上述这些改变没有发生于白色血栓组中。我们又将另外的6例白色血栓栓塞的比格犬(白色血栓+rt-PA组)进行阿替普酶(rt-PA)溶栓治疗。冠状动脉造影发现给予rt-PA溶栓后2小时,有5例出现了冠状动脉再通,再通时间为43.2±7.4分钟。白色血栓对照组未发现血管再通迹象。TTC染色显示溶栓组的心肌梗死面积明显小于非溶栓组(白色血栓组)的心肌梗死面积。结论：我们自制的白色血栓动物模型在实验方法操作上更加便利,均一性好,稳定性和成功率高。这项研究最主要的创新点在于我们利用的栓子与临床溶栓窗口内的冠状动脉血栓成分相似,都是主要由纤维蛋白构成,所以该模型可以较好地用来评价治疗ST段抬高型心肌梗死的溶栓新药疗效；该模型能够最大限度地模拟临床溶栓后心肌缺血再灌注损伤的状态,因此也可以用来探讨溶栓后心肌缺血再灌注损伤的病理变化及机制。
[Abstract]:Background: activation of the -Smad3 pathway of transforming growth factor - beta 1 (transforming growth factor- beta 1, TGF- beta 1) can aggravate myocardial ischemia reperfusion injury. The TGF- beta 1-Smad3 pathway in the myocardium of diabetic patients is obviously activated, which may be an important factor in the poor prognosis of diabetic patients undergoing myocardial ischemia reperfusion injury. One of our previous experiments has demonstrated that glutamine can reduce the myocardial damage caused by high glucose combined with hypoxia reoxygenation. The purpose of this study is to further investigate whether glutamine can play an anti diabetic myocardial ischemia-reperfusion injury by inhibiting the TGF- beta 1-Smad3 pathway. Methods: in the experiment in vitro, we cultured the H9c2 rat cardiomyocytes under the high glucose environment of 33mM, and then carried out anoxic reoxygenation and control treatment. First, the Smad3 pathway was suppressed by the specific inhibitor SIS3 of the GF- beta 1 receptor inhibitor SB431542 and Smad3, and the changes in the cell damage were observed. Secondly, we used different concentrations of the valley. The effects of aminamides on the Smad3 pathway of cardiac myocytes were observed. Finally, we used recombinant human TGF- beta 1 to activate the Smad3 pathway to verify. In vivo, we first established the diabetic rat model, and then treated normal rats and diabetic rats with saline or glutamine solution respectively. Finally, the intervention of myocardial ischemia reperfusion injury. We detected the expression level of TGF- beta 1, total Smad3, phosphorylated Smad3 and activated caspase-3 in H9c2 myocardial cells and rat myocardium by using Western blotting technique. We detected the apoptosis rate in cardiac myocytes and myocardium by TUNEL method. We also detected the large amount of apoptosis in cardiac myocytes and myocardium. Changes in myocardial infarction area and ultrastructure of myocardium under electron microscope and changes in the parameters of hemodynamic indexes of rats. Results: the results were as follows: in the experiment, the apoptosis rate of H9c2 cells in the high glucose and hypoxia reoxygenation group was the highest, and the TGF- beta 1-Smad3 pathway was also the most obvious.TGF- beta 1 receptor inhibitor in this group The specific inhibitor SIS3 of SB431542 and Smad3 can obviously downregulate the phosphorylation level of Smad3 and effectively reduce the high glucose + hypoxia reoxygenation damage to cardiac myocytes. Similarly, after supplementation of glutamine, the apoptosis level of the cells decreased and the activation of the TGF- beta 1-Smad3 pathway was inhibited. However, when exogenous TGF- was given to human recombinant TGF- After beta 1, the protective effect of glutamine was weakened and the level of cardiomyocyte apoptosis increased significantly. In the experiment of the non glutamine treatment group, the diabetic rats underwent the most severe cardiac injury after myocardial ischemia reperfusion. Mechanical indexes, which reduce myocardial apoptosis, myocardial microstructure damage and myocardial infarction area, also inhibit the activation of TGF- beta 1-Smad3 pathway in myocardial tissue. Conclusion: therefore, we conclude that the activation of TGF- beta 1-Smad3 pathway can aggravate the injury of myocardial ischemia reperfusion injury in diabetic myocardium; supplemental glutamine can be used in partial passage. Inhibition of TGF- beta 1-Smad3 pathway alleviates myocardial ischemia and reperfusion injury in diabetic myocardium. Background: in the first part of the paper, we established a model of myocardial ischemia in SD rats. The size and structure of this small rodent are different from that of the human heart, and some of the experimental applications are limited. The Beagle dog, such as the Beagle dog, has a similar heart structure and a pathophysiological reaction that is similar to human, and has a developed circulation system that is more suitable for the study of cardiovascular disease. The genetic performance of the Beagle dog is stable and excellent. It also provides protection for the homogenization of the experiment. In the second part of the paper, we first analyzed the components of coronary artery thrombosis in the clinic, and then developed a beagle myocardial ischemia model that can evaluate the therapeutic effect of thrombolytic drugs by injecting autologous embolus into the coronary artery by minimally invasive interventional technique. The experimental basis was laid for subsequent study of the mechanism of ischemic reperfusion injury after thrombolysis. Methods: We selected 18 beagle dogs, divided into 3 groups: the red thrombus group (n=6), the white thrombus group (n=6) and the white thrombus +rt-PA thrombolytic reperfusion group (n=6 only). The self-made autologous thrombus was injected into the coronary artery. The middle and far segment of the anterior descending branch of the pulse. Coronary artery embolization and thrombolytic reperfusion were monitored by electrocardiogram and electrocardiogram monitor. Coronary angiography was used to determine thrombus in body state. HE staining and electron microscopy were used to analyze different thrombus components. The area of myocardial infarction was calculated by 2,3,5- chlorination of three phenyl tetrazolium (TTC). Results: After HE staining and scanning electron microscopy, red thrombus had a loose reticular structure, and the eyes were filled with red blood cells, and the white thrombus had a tight structure. It was mainly composed of dense fibrin. Coronary angiography showed that the repass rate of the red thrombus group (autolysis) was 2/6 after 3 hours of coronary artery embolism. The repass rate of the white thrombus group was 0/6. and 2 cases of the red thrombus group had arrhythmia. The electrocardiogram showed an elevated ST segment and a decrease in the principle of the high principle T wave. These changes did not occur in the white thrombus group. We also performed the other 6 cases of white thrombus in the Beagle (white thrombus +rt-PA group). Rt-PA thrombolytic therapy. Coronary angiography was found 2 hours after rt-PA thrombolytic thrombolysis. Coronary repassage was found in 5 cases and repass time was 43.2 + 7.4 minutes. The white thrombus control group did not find the vascular repassage sign.TTC staining showed that the infarct area of the thrombolytic group was significantly smaller than that of the non thrombolytic group (white thrombus group). Death area. Conclusion: our homemade white thrombus animal model is more convenient, homogeneous, stable and successful. The main innovation of this study is that the embolus used in this study are similar to the components of coronary artery thrombus within the clinical thrombolytic window, which are mainly made up of fibrin, so this model is a model. The model can be used to evaluate the efficacy of a new thrombolytic drug for the treatment of ST segment elevation myocardial infarction. This model can simulate the state of myocardial ischemia reperfusion injury to the maximum extent, and therefore can also be used to explore the pathological changes and mechanisms of myocardial ischemia reperfusion injury after thrombolytic therapy.
【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】：2016
【分类号】：R542.2;R-332;R587.2

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