促红细胞生成素在心肌缺血动物模型中的作用及其机制研究

发布时间：2018-03-17 18:55

本文选题：促红细胞生成素　切入点：缺血性心脏病　出处：《福建医科大学》2008年博士论文　论文类型：学位论文

【摘要】： 背景动脉粥样硬化性疾病的一、二级预防已经使缺血性心脏病事件的发生率显著下降。但是缺血性心脏病依然是大多数发达国家最主要的死亡病因之一。为了进一步减少急性缺血性心脏病事件的发生率,人们仍在寻找各种更为有效的治疗方案。促红细胞生成素(erythropoietin, Epo)是由165个氨基酸组成并折叠成4个α螺旋的糖蛋白。属于I型细胞因子超家族,分子量约为30.4kDa。Epo最早运用于慢性肾功能衰竭相关性贫血治疗。此后Bernaudin等发现其在脑缺血及神经组织的机械性损伤中也发挥着保护作用,多项实验表明,脑组织局部及全身运用Epo可防治缺血诱发的神经元损伤。相似的,通过动物模型人们逐渐认识到Epo的心肌保护作用。近年来有部分小规模临床实验提示了Epo的心肌保护效应,但是其具体分子生物学机制尚未阐明。目的为了深入探讨Epo在缺血性心脏病中的生理保护作用及其临床意义。明确:1、Epo治疗是否能影响心肌细胞凋亡;2、促进血管新生、改善心肌梗塞术后心功能;3、明确EPO在缺血再灌注损伤中的保护机制,我们设计了该课题。方法 1、通过RT-PCR及Western Blot验证体外培养的心肌细胞是否表达Epo的受体。采用H_2O_2诱导体外培养心肌细胞凋亡,运用Tunel染色探讨Epo对H_2O_2诱导的心肌细胞凋亡的影响。结合细胞信号传导通路阻滞剂明确Epo抑制心肌细胞凋亡的信号传导通路。分别收集不同时间点的心肌细胞培养上清液,观测Epo对心肌细胞VEGF表达的影响。 2、采用Langendroff离体心脏缺血再灌注模型,运用不同剂量Epo观测Epo对缺血再灌注离体心脏的血流动力学影响;OCT染色观测Epo对梗塞面积的调节。结合信号传导通路阻滞剂,探讨Epo对细胞外基质代谢的调节作用及其机制。酶谱法(Zymography Assay)及大鼠缺血再灌注模型验证EPO对缺血再灌注损伤后MMPs活性的调节。结果证实心肌细胞表达Epo受体;Epo可抑制H_2O_2诱导的心肌细胞凋亡、下调心肌细胞促凋亡相关蛋白的表达,上调抗凋亡蛋白表达。Epo通过Erk及PI3K-Akt途径抑制细胞凋亡。Epo可促进心肌细胞VEGF的表达及内皮细胞增殖;通过促进心肌细胞分泌VEGF,EPO可间接对内皮细胞的增殖发生调控。利用Langendroff离体心脏缺血再灌注模型,运用不同剂量Epo证实Epo可改善缺血再灌注离体心脏的血流动力学指标;减少缺血再灌注损伤后梗塞面积。Epo激活了Jak2-Erk信号传导通路,促进了CollagenI/III的表达,抑制了MMP2及MMP9的表达。MEK拮抗剂可抑制Epo的这一保护作用。在1周的随访期中EPO显著抑制了心肌缺血再灌注损伤后MMPs的活性。结论 1、心肌细胞可表达EPO的受体,EPO经由Erk及Akt途径抑制心肌细胞凋亡; 2、EPO促进心肌细胞VEGF的合成分泌,促进内皮细胞增殖; 3、EPO治疗可显著提高心肌梗塞术后小鼠的生存率、改善心功能、抑制心脏组织凋亡、促进血管新生。 4、EPO可通过Erk途径调节心脏组织细胞外基质代谢。
[Abstract]:The background of atherosclerotic disease, two grade prevention has made the incidence of ischemic heart disease events decreased significantly. But ischemic heart disease is still one of the main causes of death in most developed countries. In order to further reduce the events of acute ischemic heart disease incidence, people are still looking for more effective treatments for erythropoietin. Erythropoietin (erythropoietin, Epo) is composed of 165 amino acids and 4 alpha helices folded into glycoproteins. Type I cytokine superfamily, a molecular weight of about 30.4kDa.Epo was first used in the treatment of chronic renal failure associated with anemia. Then Bernaudin found to play in cerebral ischemia and neural tissue machine injury protection, a number of experiments showed that the neuronal damage in brain tissue of local and systemic use of Epo can prevent ischemia induced by animals. Similar. Models have gradually recognized the protective effect of Epo on myocardium. In recent years, some small-scale clinical trials have suggested the myocardial protective effect of Epo, but the specific molecular biological mechanisms have not yet been elucidated.
Objective to investigate the physiological protective effect of Epo in ischemic heart disease and its clinical significance. Clear: 1, Epo treatment can influence the apoptosis of myocardial cells; 2, promote angiogenesis and improve cardiac function after myocardial infarction; 3, clear EPO in ischemia reperfusion injury protection mechanism, we designed this topic.
Method
1, whether through RT-PCR and Western Blot to verify the in vitro cultured myocardial cells. The expression of Epo receptor induced by H_2O_2 cultured myocardial cells apoptosis in vitro, the use of Tunel to explore the effects of Epo on myocardial cell apoptosis induced by H_2O_2 staining. The signal transduction pathway combined with cell signaling pathway blocker clear Epo inhibition of myocardial cell apoptosis were collected from different. At the time of the myocardial cell culture supernatant, observing the effects of Epo on the expression of VEGF in myocardial cell.
2, using Langendroff isolated heart ischemia reperfusion model with different doses of Epo Epo observation on ischemia reperfusion hemodynamics from body cardiac effects; OCT staining observation of Epo regulation on infarction area. Combined with the signal transduction pathway blocker, to investigate the regulatory effect of Epo on extracellular matrix metabolism enzymes (method and its mechanism. Zymography Assay) and the regulation of ischemia reperfusion rat model validation EPO on ischemia reperfusion injury of MMPs activity.
The results confirmed that the expression of Epo receptor in myocardial cells; myocardial Epo can inhibit cell apoptosis induced by H_2O_2, expression of myocardial cell apoptosis related protein, up regulation of anti apoptotic protein.Epo expression through Erk and PI3K-Akt pathway to inhibit apoptosis and expression of.Epo can promote the proliferation of endothelial cells of myocardial cell VEGF; myocardial cells by promoting the secretion of VEGF, EPO the indirect control of the proliferation of endothelial cells. Using Langendroff isolated heart ischemia reperfusion model with different doses of Epo showed that Epo can improve the hemodynamics index from ischemia reperfusion cardiac ischemia reperfusion injury; reduce infarct volume after.Epo activation of the Jak2-Erk signaling pathway, promote the expression of CollagenI/III, this protective effect inhibited the expression of.MEK antagonist MMP2 and MMP9 inhibited Epo. During the follow-up period of 1 weeks in EPO significantly inhibited the myocardial ischemia reperfusion The activity of MMPs after injury.
conclusion
1, cardiac myocytes can express EPO receptor, and EPO inhibits cardiomyocyte apoptosis via Erk and Akt pathway.
2, EPO promotes the synthesis and secretion of VEGF in cardiac myocytes and promotes the proliferation of endothelial cells.
3, EPO therapy can significantly improve the survival rate of mice after myocardial infarction, improve cardiac function, inhibit cardiac apoptosis and promote angiogenesis.
4, EPO can regulate the metabolism of extracellular matrix in the cardiac tissue by Erk pathway.

【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R-332;R54

【引证文献】