發(fā)布時(shí)間：2019-05-23 01:41

【摘要】：一、研究背景心肌慢性缺氧是臨床諸多疾病的共同病理生理過(guò)程,可見于紫紺型先心病、肺源性心臟病以及高原性心臟病等全身系統(tǒng)性缺氧疾病,也常見于冠狀動(dòng)脈粥樣硬化、心肌肥厚等因素所導(dǎo)致的心肌局部缺血缺氧狀態(tài)。研究慢性缺氧心肌的適應(yīng)性反應(yīng)及其機(jī)制,對(duì)上述臨床疾病的治療具有重要意義。自噬是細(xì)胞內(nèi)高度保守的分解代謝過(guò)程,基礎(chǔ)水平的自噬有助于胞內(nèi)蛋白和細(xì)胞器的更新以及細(xì)胞穩(wěn)態(tài)的維持;但在營(yíng)養(yǎng)缺乏、氧化應(yīng)激等因素刺激下,自噬水平增高,從而降解多余蛋白以提供小分子代謝底物,同時(shí)也能清除受損細(xì)胞器從而避免細(xì)胞損傷。既往研究發(fā)現(xiàn),在TAC誘導(dǎo)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;在來(lái)源于紫紺型先心病患者的手術(shù)標(biāo)本中,檢測(cè)發(fā)現(xiàn)自噬增強(qiáng)。自噬與心血管疾病的發(fā)生、發(fā)展存在密切聯(lián)系,研究慢性缺氧心肌細(xì)胞自噬的變化及其機(jī)制,可能為治療該類疾病提供潛在的干預(yù)靶點(diǎn)。AMPK是細(xì)胞內(nèi)保守的絲/蘇氨酸激酶,被認(rèn)為是細(xì)胞內(nèi)能量穩(wěn)態(tài)調(diào)控的關(guān)鍵分子。當(dāng)細(xì)胞內(nèi)ATP含量減少時(shí),AMPK被激活,抑制合成代謝,促進(jìn)分解代謝和ATP生成。AMPK也是自噬通路的上游分子,在細(xì)胞應(yīng)激時(shí),AMPK的激活也參與了自噬通路的調(diào)控。研究發(fā)現(xiàn),AMPK的激活可以促進(jìn)慢性缺氧心肌細(xì)胞的適應(yīng)與存活,但其具體機(jī)制仍有待進(jìn)一步闡明。AMPK分子由α、β和γ三個(gè)亞基構(gòu)成,α為催化亞基,負(fù)責(zé)激活的AMPK分子的功能執(zhí)行,存在α1與α2兩種亞型,α2亞型在心臟分布較多。本課題研究中,我們首先建立小鼠的慢性缺氧模型,檢測(cè)慢性缺氧對(duì)小鼠心肌細(xì)胞自噬水平的影響,然后采用AMPKα2基因敲除的小鼠,觀察其對(duì)常氧或缺氧條件下小鼠心肌細(xì)胞自噬水平以及小鼠生存曲線的影響。二、研究方法1.小鼠慢性缺氧模型的建立:參照專利設(shè)計(jì),制作一簡(jiǎn)易小動(dòng)物常壓低氧艙,依據(jù)文獻(xiàn)資料,將小鼠放置于10%的低氧艙內(nèi)飼養(yǎng)4周,建立慢性缺氧模型,并通過(guò)檢測(cè)紅細(xì)胞等相關(guān)指標(biāo),證實(shí)建模成功。2.慢性缺氧對(duì)小鼠心肌細(xì)胞自噬水平的影響:取野生型C57小鼠隨機(jī)分為常氧組和缺氧組,通過(guò)Western blot檢測(cè)小鼠心肌LC3-Ⅱ/LC3-Ⅰ比值及p62表達(dá)的變化,用免疫熒光染色法檢測(cè)小鼠心肌冰凍切片中LC3的變化。3.AMPKα2敲除對(duì)小鼠心肌細(xì)胞自噬的影響:采用AMPKα2+/-雜合子小鼠配對(duì)繁殖,后代小鼠行基因型鑒定,將野生型與AMPKα2-/-小鼠分別分為常氧組和缺氧組,即:野生型常氧組、野生型缺氧組、敲除型常氧組和敲除型缺氧組,共4組(每組n=10)。通過(guò)Western blot檢測(cè)小鼠心肌細(xì)胞LC3-Ⅱ/LC3-Ⅰ比值變化及免疫熒光染色法檢測(cè)小鼠心肌細(xì)胞LC3表達(dá)的變化來(lái)反映自噬的改變,記錄小鼠死亡的時(shí)間與例數(shù),繪制小鼠生存曲線。三、研究結(jié)果1.慢性缺氧模型的建立:和常氧組比較,缺氧組小鼠的紅細(xì)胞與血紅蛋白均顯著增加(P0.05);日均進(jìn)食量與體重均顯著降低(P0.05);心臟/體重比值與雙肺/體重比值均顯著下降(P0.05)。2.慢性缺氧對(duì)小鼠心肌細(xì)胞自噬水平的影響:和常氧組比較,Western blot顯示缺氧組小鼠心肌組織LC3-Ⅱ/LC3-Ⅰ比值顯著升高(P0.05),p62水平顯著降低(P0.05);免疫熒光染色顯示其冰凍切片中LC3表達(dá)明顯增加。3.AMPKα2敲除對(duì)小鼠心肌細(xì)胞自噬的影響:常氧條件下,AMPKα2-/-小鼠心肌細(xì)胞LC3-Ⅱ/LC3-Ⅰ比值較野生型無(wú)顯著統(tǒng)計(jì)學(xué)差異(P0.05);但缺氧條件下,AMPKα2-/-小鼠心肌細(xì)胞LC3-Ⅱ/LC3-Ⅰ比值較野生型有顯著下降(P0.05),免疫熒光染色結(jié)果與Western blot結(jié)果一致。缺氧條件下,AMPKα2-/-小鼠中位生存時(shí)間明顯縮短(P0.05)。4.統(tǒng)計(jì)分析:使用SPSS13.0進(jìn)行統(tǒng)計(jì)分析,兩組均數(shù)比較采用t檢驗(yàn),多組(≥3)均數(shù)比較采用單因素方差分析。四、主要結(jié)論1.慢性缺氧條件下,野生型小鼠心肌細(xì)胞自噬水平增高,說(shuō)明自噬可能是心肌細(xì)胞對(duì)慢性缺氧的適應(yīng)性性機(jī)制之一。2.與野生型小鼠相比,在常氧條件下,AMPKα2敲除小鼠心肌細(xì)胞自噬水平無(wú)明顯變化,但在慢性缺氧條件下,AMPKα2敲除小鼠心肌細(xì)胞自噬水平顯著降低,中位生存時(shí)間顯著較短,提示AMPKα2所介導(dǎo)的自噬可能在心肌慢性缺氧的適應(yīng)中具有重要意義。
[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，

本文編號(hào)：2483481