AMPKα2调节自噬参与心肌慢性缺氧适应的研究
发布时间：2019-05-23 01:41
【摘要】：一、研究背景心肌慢性缺氧是临床诸多疾病的共同病理生理过程,可见于紫绀型先心病、肺源性心脏病以及高原性心脏病等全身系统性缺氧疾病,也常见于冠状动脉粥样硬化、心肌肥厚等因素所导致的心肌局部缺血缺氧状态。研究慢性缺氧心肌的适应性反应及其机制,对上述临床疾病的治疗具有重要意义。自噬是细胞内高度保守的分解代谢过程,基础水平的自噬有助于胞内蛋白和细胞器的更新以及细胞稳态的维持;但在营养缺乏、氧化应激等因素刺激下,自噬水平增高,从而降解多余蛋白以提供小分子代谢底物,同时也能清除受损细胞器从而避免细胞损伤。既往研究发现,在TAC诱导的小鼠心肌肥厚模型及心肌缺血/再灌注模型中,自噬水平增高;在来源于紫绀型先心病患者的手术标本中,检测发现自噬增强。自噬与心血管疾病的发生、发展存在密切联系,研究慢性缺氧心肌细胞自噬的变化及其机制,可能为治疗该类疾病提供潜在的干预靶点。AMPK是细胞内保守的丝/苏氨酸激酶,被认为是细胞内能量稳态调控的关键分子。当细胞内ATP含量减少时,AMPK被激活,抑制合成代谢,促进分解代谢和ATP生成。AMPK也是自噬通路的上游分子,在细胞应激时,AMPK的激活也参与了自噬通路的调控。研究发现,AMPK的激活可以促进慢性缺氧心肌细胞的适应与存活,但其具体机制仍有待进一步阐明。AMPK分子由α、β和γ三个亚基构成,α为催化亚基,负责激活的AMPK分子的功能执行,存在α1与α2两种亚型,α2亚型在心脏分布较多。本课题研究中,我们首先建立小鼠的慢性缺氧模型,检测慢性缺氧对小鼠心肌细胞自噬水平的影响,然后采用AMPKα2基因敲除的小鼠,观察其对常氧或缺氧条件下小鼠心肌细胞自噬水平以及小鼠生存曲线的影响。二、研究方法1.小鼠慢性缺氧模型的建立:参照专利设计,制作一简易小动物常压低氧舱,依据文献资料,将小鼠放置于10%的低氧舱内饲养4周,建立慢性缺氧模型,并通过检测红细胞等相关指标,证实建模成功。2.慢性缺氧对小鼠心肌细胞自噬水平的影响:取野生型C57小鼠随机分为常氧组和缺氧组,通过Western blot检测小鼠心肌LC3-Ⅱ/LC3-Ⅰ比值及p62表达的变化,用免疫荧光染色法检测小鼠心肌冰冻切片中LC3的变化。3.AMPKα2敲除对小鼠心肌细胞自噬的影响:采用AMPKα2+/-杂合子小鼠配对繁殖,后代小鼠行基因型鉴定,将野生型与AMPKα2-/-小鼠分别分为常氧组和缺氧组,即:野生型常氧组、野生型缺氧组、敲除型常氧组和敲除型缺氧组,共4组(每组n=10)。通过Western blot检测小鼠心肌细胞LC3-Ⅱ/LC3-Ⅰ比值变化及免疫荧光染色法检测小鼠心肌细胞LC3表达的变化来反映自噬的改变,记录小鼠死亡的时间与例数,绘制小鼠生存曲线。三、研究结果1.慢性缺氧模型的建立:和常氧组比较,缺氧组小鼠的红细胞与血红蛋白均显著增加(P0.05);日均进食量与体重均显著降低(P0.05);心脏/体重比值与双肺/体重比值均显著下降(P0.05)。2.慢性缺氧对小鼠心肌细胞自噬水平的影响:和常氧组比较,Western blot显示缺氧组小鼠心肌组织LC3-Ⅱ/LC3-Ⅰ比值显著升高(P0.05),p62水平显著降低(P0.05);免疫荧光染色显示其冰冻切片中LC3表达明显增加。3.AMPKα2敲除对小鼠心肌细胞自噬的影响:常氧条件下,AMPKα2-/-小鼠心肌细胞LC3-Ⅱ/LC3-Ⅰ比值较野生型无显著统计学差异(P0.05);但缺氧条件下,AMPKα2-/-小鼠心肌细胞LC3-Ⅱ/LC3-Ⅰ比值较野生型有显著下降(P0.05),免疫荧光染色结果与Western blot结果一致。缺氧条件下,AMPKα2-/-小鼠中位生存时间明显缩短(P0.05)。4.统计分析:使用SPSS13.0进行统计分析,两组均数比较采用t检验,多组(≥3)均数比较采用单因素方差分析。四、主要结论1.慢性缺氧条件下,野生型小鼠心肌细胞自噬水平增高,说明自噬可能是心肌细胞对慢性缺氧的适应性性机制之一。2.与野生型小鼠相比,在常氧条件下,AMPKα2敲除小鼠心肌细胞自噬水平无明显变化,但在慢性缺氧条件下,AMPKα2敲除小鼠心肌细胞自噬水平显著降低,中位生存时间显著较短,提示AMPKα2所介导的自噬可能在心肌慢性缺氧的适应中具有重要意义。
[Abstract]:I. The study of chronic hypoxia in the background of myocardial infarction is a common pathophysiological process of many diseases, and it can be found in the systemic and systemic hypoxia diseases such as the congenital heart disease, the pulmonary heart disease and the high altitude heart disease. It is also common in the coronary atherosclerosis. The myocardial ischemia-deficient state caused by the factors such as myocardial hypertrophy. To study the adaptive response of chronic hypoxia and its mechanism, it is of great significance to the treatment of the above-mentioned clinical diseases. autophagy is a highly conserved catabolic process in the cell, the autophagy of the basal level contributes to the renewal of the intracellular proteins and organelles and the maintenance of the steady state of the cells; however, the autophagy level is increased under the stimulation of the nutritional deficiency, oxidative stress, and the like, Thereby degrading the excess protein to provide a small molecular metabolic substrate while also removing the damaged organelles to avoid cell damage. The previous study found that the level of autophagy in the model of cardiac hypertrophy and myocardial ischemia/ reperfusion induced by TAC was increased; in the surgical specimens from patients with congenital heart disease, autophagy was detected. Since autophagy is closely related to the occurrence and development of cardiovascular diseases, it is possible to provide a potential intervention target for the treatment of such diseases. AMPK is a conserved filament/ threonine kinase in the cell and is thought to be a key molecule for steady-state regulation of intracellular energy. When the intracellular ATP content is reduced, AMPK is activated to inhibit the synthesis of metabolism, promote catabolism and ATP production. AMPK is also an upstream molecule of autophagy, and the activation of AMPK is involved in the regulation of autophagy. It is found that the activation of AMPK can promote the adaptation and survival of chronic hypoxic myocardial cells, but its specific mechanism is still to be clarified. AMPK (AMPK) is composed of three subunits, which is a catalytic subunit, which is responsible for the function of the activated AMPK molecule. In this study, we first set up the chronic hypoxia model of the mouse, detect the effect of chronic hypoxia on the autophagy level of the myocardial cells of the mouse, and then use the AMPK-2 gene to knock out the mouse, The effects of their normal oxygen or hypoxia on the autophagy and the survival curves of the mice were observed. II. Study Method 1. The establishment of chronic hypoxia model in mice: a simple small animal was made to lower the oxygen cabin with reference to the design of the patent. According to the literature, the mice were placed in 10% of the hypoxic chamber for 4 weeks, and the chronic hypoxia model was established, and the correlation indexes such as red blood cells were tested to confirm that the modeling was successful. The effect of chronic hypoxia on the level of autophagy in the myocyte of mice: the wild-type C57 mice were randomly divided into the normal oxygen group and the hypoxia group, and the changes of the expression of the LC3-鈪，
本文编号：2483481
[Abstract]:I. The study of chronic hypoxia in the background of myocardial infarction is a common pathophysiological process of many diseases, and it can be found in the systemic and systemic hypoxia diseases such as the congenital heart disease, the pulmonary heart disease and the high altitude heart disease. It is also common in the coronary atherosclerosis. The myocardial ischemia-deficient state caused by the factors such as myocardial hypertrophy. To study the adaptive response of chronic hypoxia and its mechanism, it is of great significance to the treatment of the above-mentioned clinical diseases. autophagy is a highly conserved catabolic process in the cell, the autophagy of the basal level contributes to the renewal of the intracellular proteins and organelles and the maintenance of the steady state of the cells; however, the autophagy level is increased under the stimulation of the nutritional deficiency, oxidative stress, and the like, Thereby degrading the excess protein to provide a small molecular metabolic substrate while also removing the damaged organelles to avoid cell damage. The previous study found that the level of autophagy in the model of cardiac hypertrophy and myocardial ischemia/ reperfusion induced by TAC was increased; in the surgical specimens from patients with congenital heart disease, autophagy was detected. Since autophagy is closely related to the occurrence and development of cardiovascular diseases, it is possible to provide a potential intervention target for the treatment of such diseases. AMPK is a conserved filament/ threonine kinase in the cell and is thought to be a key molecule for steady-state regulation of intracellular energy. When the intracellular ATP content is reduced, AMPK is activated to inhibit the synthesis of metabolism, promote catabolism and ATP production. AMPK is also an upstream molecule of autophagy, and the activation of AMPK is involved in the regulation of autophagy. It is found that the activation of AMPK can promote the adaptation and survival of chronic hypoxic myocardial cells, but its specific mechanism is still to be clarified. AMPK (AMPK) is composed of three subunits, which is a catalytic subunit, which is responsible for the function of the activated AMPK molecule. In this study, we first set up the chronic hypoxia model of the mouse, detect the effect of chronic hypoxia on the autophagy level of the myocardial cells of the mouse, and then use the AMPK-2 gene to knock out the mouse, The effects of their normal oxygen or hypoxia on the autophagy and the survival curves of the mice were observed. II. Study Method 1. The establishment of chronic hypoxia model in mice: a simple small animal was made to lower the oxygen cabin with reference to the design of the patent. According to the literature, the mice were placed in 10% of the hypoxic chamber for 4 weeks, and the chronic hypoxia model was established, and the correlation indexes such as red blood cells were tested to confirm that the modeling was successful. The effect of chronic hypoxia on the level of autophagy in the myocyte of mice: the wild-type C57 mice were randomly divided into the normal oxygen group and the hypoxia group, and the changes of the expression of the LC3-鈪，
本文编号：2483481
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