缺血/缺氧对心脏胰岛素样生长因子2受体表达调控的实验研究

【作者】 张秀芳；

【导师】 魏英杰； 周建业； 张浩；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 尽管心血管疾病的治疗方法现已取得了巨大的进展,但是各种心脏疾病导致的心力衰竭在全球仍然具有很高的发病率和死亡率。心力衰竭是一种由多因素引起的疾病,内在的分子机制还不是很清楚,但很可能与潜在的基因和蛋白质表达改变有关。本实验室已经证明胰岛素样生长因子2受体(insulin-like growth factor 2 receptor, IGF-2R)在心衰患者心脏组织的表达显著提高,提示IGF-2R可能在心力衰竭的发生中起重要的作用。本实验检测了IGF-2R在大鼠心肌梗死不同时间点后的动态变化规律,并在体外以缺氧无血清模拟心肌缺血缺氧环境,研究IGF-2R在心肌细胞缺氧无血清时表达的变化以及其与细胞凋亡的关系,为阐明IGF-2R在心肌梗死后心力衰竭发生发展过程中的作用奠定基础。本论文主要包括以下研究内容：1雄性SD大鼠180只随机分为两组：心肌梗死模型组和假手术对照组。通过结扎大鼠左冠状动脉前降支制作大鼠心梗模型,梗死后1天、3天、7天、2周、4周和8周取大鼠左心室组织,采用酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)检测大鼠心肌梗死区IGF-2R的表达变化规律。结果表明,大鼠心肌梗死后1天、3天IGF-2R表达下降,随后逐渐恢复至正常。2体外模拟心肌缺血缺氧环境,观察IGF-2R在心肌细胞缺氧无血清时表达的变化。离体培养SD乳鼠心肌细胞,缺氧无血清处理不同时间点后收获细胞,应用反转录聚合酶链式反应(RT-PCR)和实时定量RT-PCR(real time RT-PCR)测定IGF-2R mRNA水平的表达变化,并进一步应用ELISA方法检测IGF-2R在蛋白水平的改变。与对照组相比,IGF-2R mRNA在缺氧无血清处理12h和24h后表达显著增高,缺氧无血清24h后IGF-2R蛋白表达也升高。3采用脂质体介导siRNA转染心肌细胞抑制IGF-2R mRNA的表达后,采用流式细胞仪检测IGF-2R对缺氧无血清引起的心肌细胞凋亡的影响。结果表明,阻断IGF-2R mRNA的表达对缺氧无血清所引起的心肌细胞凋亡影响不明显。更多还原

【Abstract】 Despite considerable advances in the treatment of cardiovascular disease, it remains the leading cause of mortality and morbility in the world. Underlying molecular causes of cardiac dysfunction in most heart diseases are still largely unknown but are expected to result from causal alterations in gene and protein expression. We had already found IGF-2R was highly expressed in the tissues samples from heart failure patients compaired with non-failing hearts. In this article we studied changes of expression of IGF-2R during ventricular remodeling following myocardial infarction. In addition, we studied weather IGF-2R played important roles in the process of ischemia induced apoptosis. All of these studies will contribute to the new strategies of treatment of heart falure.Main contents of this study including:1 Myocardial infarction was created in SD rats by ligation of the left anterior descending coronary artery. The rats were randomly divided into 2 groups:sham group and myocardial infarction (MI) group. Animals were sacrificed at 1day,3day,7days, 2weeks,4weeks,8weeks after operation and the left ventricular tissues were isolated for determination of IGF-2R by enzyme-linked immunosorbent assay (ELISA). Compaired with the sham group, IGF-2R protein was downregulated 1 and 3day after MI, and then returned to normal levels.2 Cultured neonatal rat cardiomyocytes were randomly divided into control group and hypoxia and serum deprivation (H/SD) group. Expression of IGF-2R mRNA was measured by RT-PCR and real time RT-PCR. Level of IGF-2R protein was measured by ELISA.Compared with control group, the expression of IGF-2R mRNA increased significantly (P<0.01) in cardiomyocytes cultured in H/SD condition for 12h and 24h, and IGF-2R protein was upregulated in cardiomyocytes cultured in H/SD condition for 24h(P<0.05). H/SD could induce IGF-2R expression, and IGF-2R may involve in the mechanism of cardiomyocyte damage induced by ischemia.3 Cultured neonatal rat cardiomyocytes were randomly divided into control group, H/SD group, scramble siRNA group and IGF-2R siRNA group.Percentage of apoptotic cells measured by flow cytometry.The results demonstrated that H/SD induced apoptosis of cardiomyocytes, but we did not found that down-regulation of IGF-2R mRNA reduced apotosis in neonatal rat cardiomyocytes cultured in H/SD condition.更多还原