【作者】 刘亚忠；

【导师】 董士民；

【作者基本信息】 河北医科大学 ， 内科学， 2010， 硕士

【摘要】 目的:人们往往使用再灌注疗法恢复缺血心肌细胞的血流供应。心肌细胞凋亡在心肌缺血再灌注损伤中普遍存在,并且是缺血再灌注早期心肌细胞死亡的主要方式。在心肌缺血再灌注损伤发病机制中,细胞凋亡可能是再灌注损伤的重要组成部分之一,而且凋亡在特定阶段前的干预部分是可逆的。研究再灌注损伤时心肌细胞抗凋亡机制可以提供一个潜在的方法来减轻再灌注诱导的细胞凋亡。近年来,人们认识到重组人促红细胞生成素(recombinant human erythropoietin, EPO)在心血管疾病中的重要作用。Calvillo等在培养的大鼠心肌细胞缺氧损伤实验中发现,应用EPO的细胞凋亡率降低50%,证实EPO具有抗心肌细胞凋亡的作用。但是EPO对凋亡基因的研究尚不太清楚。Bcl-2蛋白基因家族是控制凋亡的关键基因,包括抗凋亡基因Bcl-2和促凋亡基因Bax,但是EPO是否通过Bcl-2、Bax发挥抗凋亡作用尚不清楚,研究Bcl-2蛋白基因家族有助于进一步了解EPO的抗凋亡作用。研究发现血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达,可能使血管新生更容易,远期可以减少心肌细胞的凋亡。VEGF产生的这种效应的机制也可能是升高抗凋亡蛋白Bcl-2的表达,降低促凋亡蛋白Bax的表达,从而减少细胞凋亡。因此,EPO可能是通过影响Bcl-2、Bax蛋白及基因的表达,从而抑制细胞凋亡,达到改善急性心肌缺血再灌注后心脏功能的作用。本实验通过建立大鼠心肌缺血再灌注模型,应用EPO观察Bcl-2蛋白,Bax蛋白,VEGF蛋白表达及Bcl-2mRNA、BaxmRNA表达,来探讨促红细胞生成素的心脏保护作用。方法:健康雄性Sprague Dawley大鼠54只,清洁级,12-15周龄,体重210-260g,平均225g,阻断大鼠左冠状动脉前降支建立大鼠急性心肌缺血再灌注模型,随机分为3组,假手术组(Sham组)、缺血再灌注对照组(IR组)和EPO组(EPO,EPO 3000U/kg/d共3天)。应用免疫组化方法测定Bcl-2、Bax蛋白及VEGF蛋白表达,应用RT-PCR方法测定Bcl-2mRNA、BaxmRNA。数据采用SPSS 17.0统计软件处理,组间比较采用单因素方差分析,两两比较采用q检验。结果:(1)EPO对大鼠边缘区心肌细胞Bcl-2蛋白表达的影响:与Sham组相比较,IR组、EPO组Bcl-2蛋白表达在48h、2w、3w均显著降低,有统计学意义。与IR组相比较,EPO组Bcl-2蛋白表达在48h、2w、3w均增高,分别增高27.7%、31.4%、25.9%(均P<0.01)。(2)EPO对大鼠边缘区心肌细胞Bax蛋白表达的影响:与Sham组相比较,IR组、EPO组Bax蛋白表达在48h、2w、3w均显著增高,有统计学意义。与IR组相比较,EPO组Bax蛋白表达在48h、2w、3w均降低,分别降低20.9%、18.0%、16.7%(均P<0.01)。(3)EPO对大鼠边缘区心肌细胞Bcl-2/Bax蛋白表达的影响:与Sham组相比较,IR组、EPO组Bcl-2/Bax比值在各时间点均显著降低(均P<0.05)。与IR组相比较,EPO组Bcl-2/Bax比值在各时间点均显著增高(均P<0.05)。(4)EPO对大鼠边缘区心肌细胞Bcl-2mRNA表达的影响:与Sham组相比较,IR组、EPO组Bcl-2mRNA表达在48h、2w、3w均显著降低,有统计学意义。与IR组相比较,EPO组Bcl-2mRNA表达在48h、2w、3w均增高,分别增高61.0%、32.7%、39.9%(P<0.01)。(5)EPO对大鼠边缘区心肌细胞表达的影响:与Sham组相比较,BaxmRNA、EPO组BaxmRNA表达在48h、2w、3w均显著增高,有统计学意义。与IR组相比较,EPO组BaxmRNA表达在48h、2w、3w均降低,分别降低11.4%、7.7%、34.2%(P<0.01)。(6)EPO对大鼠边缘区心肌细胞Bcl-2/BaxmRNA表达的影响:与Sham组相比较,IR组、EPO组Bcl-2/BaxmRNA比值在各时间点均显著降低(P<0.05)。与IR组相比较,EPO组Bcl-2/BaxmRNA比值在各时间点均显著增高( P<0.05)。(7)EPO对大鼠边缘区心肌细胞VEGF蛋白表达的影响:与Sham组相比较,IR组、EPO组VEGF蛋白表达在48h、2w、3w均显著增高,有统计学意义。与IR组相比较,EPO组VEGF蛋白表达在48h、2w、3w均增高,分别增高22.1%、21.4%、20.5%(P<0.01 or P<0.05)。结论:(1)大鼠急性心肌缺血再灌注后边缘区心肌细胞Bcl-2蛋白及mRNA的低表达,Bax蛋白及mRNA的高表达。(2)EPO在大鼠缺血再灌注后可增加边缘区心肌Bcl-2蛋白、VEGF蛋白及mRNA的表达,降低Bax蛋白及mRNA的表达,增高Bcl-2/Bax的比值。表明了EPO可能通过调节Bcl-2、Bax的表达而抑制缺血再灌注损伤,减少细胞凋亡,产生对心脏的保护作用。更多还原

【Abstract】 Objective: It is often used reperfusion therapy to recover the blood supply of the ischaemic myocardial cell. Myocardial cell apoptosis in myocardial ischemia-reperfusion injury is very common, and apoptosis is the main deadly forms in the early stage of ischemia-reperfusion. Studying the anti-apoptosis mechanisms after ischemia-reperfusion could provide a potential way to reduce the apoptosis induced by reperfusion.In recent years, it is recognized the important role of EPO(recombinant human erythropoietin) in cardiovascular diseases. Calvillo found that adding EPO in cultured hypoxic injury rat myocardial cells, apoptosis decreased by 50% and confirmed that EPO has the anti- apoptosis function. But the EPO on apoptosis genes is less clear. Bcl-2 protein gene family is critical in apoptosis genes, include anti-apoptosis gene Bcl-2 and pro-apoptosis gene Bax,but it is not clear that the anti-apoptosis function of EPO is created via Bcl-2 protein gene family, so studying the Bcl-2 protein gene family is helpful for explaining the protecting function of EPO. Study found that the expression of VEGF (vascular endothelial growth factor) may make it easier to angiogenesis, and can reduce long term myocardial cell apoptosis. The mechanisms of VEGF may increase the expression of anti-apoptotic protein Bcl-2, reduce the expression of pro-apoptotic protein Bax, thereby reducing apoptosis. So EPO inhibit apoptosis may be by influencing Bcl-2 protein family, and then improve the cardiac function after acute myocardial ischemia-reperfusion.In this study, a animal model of IR(ischemia-reperfusion) was used, and followed treatment with recombinant human EPO (EPO). The expression of Bcl-2 protein, Bax protein, VEGF protein, Bcl-2 mRNA, Bax mRNA were obtained after IR, and then to investigate the protective mechanismss of EPO. Methods: 54 healthy male Sprague Dawley rats (12-15 weeks and 210-260g) were recruited. Rat models of IR were induced by blocking left anterior decending coronary artery. The 54 rats were divided into 3 groups randomly: sham-operated group, IR group and EPO group (recombinant human EPO 3000U·kg-1·d-1 intraperitoneal injection, for 3 days). The Bcl-2 protein, Bax protein, VEGF protein, were quantified by immunohistochemical method. RT-PCR was used to observe the gene expression of Bcl-2 mRNA, Bax mRNA. All data were analyzed with SPSS version 17.0 statistical software. The comparison among groups was analyzed by One-Way ANOVA and S-N-K test.Results: (1) The effect of EPO on the expression of Bcl-2 protein in border areas: Compared with sham-operated group, the expression of Bcl-2 protein detected in border areas significantly decreased in IR group and EPO group at 48h, 2w and 3w (P<0.01); Compared with IR group, the expression of Bcl-2 protein detected in border areas raised 27.7%, 31.4%, 25.9% respectively in EPO group at 48h, 2w and 3w (P<0.01). (2) The effect of EPO on the expression of Bax protein in border areas: Compared with sham-operated group, the expression of Bax protein detected in border areas significantly increased raised in IR group and EPO group at 48h(P<0.01); Compared with IR group, the expression of Bax protein detected in border areas lowered 20.9%, 18.0%, 16.7% respectively in EPO group at 48h, 2w and 3w (P<0.01). (3) The effect of EPO on the ratio of Bcl-2 and Bax protein in border areas: Compared with sham-operated group, the ratio of Bcl-2 and Bax protein was significantly decreased in IR group and EPO group at the different time (P<0.05); Compared with IR group, the ratio of Bcl-2 and Bax protein was in EPO group at the different time (P<0.05); (4) The effect of EPO on the expression of Bcl-2mRNA in border areas: Compared with sham-operated group, the expression of Bcl-2mRNA detected in border areas decreased in IR group and EPO group at 48h, 2w and 3w (P<0.01); Compared with IR group, the expression of Bcl-2mRNA detected in border areas raised 61.0%, 32.7%, 39.9% respectively in EPO group at 48h, 2w and 3w (P<0.01). (5) The effect of EPO on the expression of BaxmRNA in border areas: Compared with sham-operated group, the expression of BaxmRNA detected in border areas significantly increased in IR group and EPO group at 48h, 2w and 3w (P<0.01); Compared with IR group, the expression of BaxmRNA detected in border areas lowered 11.4%, 7.7%, 34.2% respectively in EPO group at 48h, 2w and 3w (P<0.01).(6) The effect of EPO on the ratio of Bcl-2 and Bax mRNA in border areas: Compared with sham-operated group, the ratio of Bcl-2 and Bax mRNA was significantly decreased in IR group and EPO group at the different time (P<0.05); Compared with IR group, the ratio of Bcl-2 and Bax mRNA was significantly increased in EPO group at the different time (P<0.05); (7) The effect of EPO on the expression of VEGF protein in border areas: Compared with sham-operated group, the expression of VEGF protein detected in border areas significantly increased in IR group and EPO group at 48h, 2w and 3w; (P<0.01 or P<0.05); Compared with IR group, the expression of VEGF protein detected in border areas raised 22.1%, 21.4%, 20.5% respectively in EPO group at 48h, 2w and 3w (P<0.01).Conculsion:(1) This study showed that the lower expression of Bcl-2 protein and mRNA, higher expression of Bax protein and mRNA in border areas after myocardial ischemia-reperfusion. (2) EPO could increase the expression of Bcl-2 protein ,VEGF protein and Bcl-2 mRNA, reduce the expression of Bax protein and Bax mRNA, increase the ratio of Bcl-2/Bax in border areas after myocardial ischemia-reperfusion. It is possible that EPO may bring cardiac protective function by regulateing the expression of Bcl-2 and Bax to inhibit the ischemia-reperfusion injury.更多还原