本文選題：自噬 + 乙醇脫氫酶�。� 參考：《山東大學(xué)》2016年博士論文

【摘要】：研究背景正常的心臟功能是保證人類健康與生活質(zhì)量的基本條件。但心肌細(xì)胞屬于永久性細(xì)胞,在分化完成后幾乎不可再生。因此,維持正常的心肌細(xì)胞功能十分重要。然而,心肌細(xì)胞功能的維持面臨很多挑戰(zhàn)�；蚨鄳B(tài)性對心臟功能有先天性的影響。直接編碼心臟發(fā)育相關(guān)蛋白的基因發(fā)生異�？蓪�(dǎo)致心臟畸形及心臟功能嚴(yán)重受損。但在健康人群中,即使年齡、性別相同,體重與身高也類似的人心臟功能依然有差異,尤其是當(dāng)心臟功能面臨一定的應(yīng)激挑戰(zhàn)時,這種差異尤為明顯。這歸因于人類的基因多態(tài)性。本文將要討論酒精代謝酶乙醇脫氫酶(Alcohol dehydrogenase, ADH)與巨噬細(xì)胞遷移抑制因子(Macrophage migration inhibitory factor, MIF)的轉(zhuǎn)基因或基因敲除模型對心臟功能的影響。ADH將酒精轉(zhuǎn)化為乙醛,完成酒精代謝的第一步。ADH1作為ADH的一種亞型中在酒精代謝中起最主要作用。人類編碼ADH1亞結(jié)構(gòu)ADH1B的基因存在基因多態(tài)性。突變型ADH1B攜帶者顯示出超強的ADH酶活性。在東亞人群中,ADH1B的突變率為40%-90%,遠(yuǎn)高于非洲與歐洲人群。基礎(chǔ)研究中,ADH過表達(dá)模型常用于研究酒精代謝對心臟功能的影響,但ADH蛋白本身對心臟功能的影響只有較少研究,且仍存在爭議。研究ADH多態(tài)性對心臟功能的影響對東亞人群具有重要意義。MIF是T細(xì)胞分泌的介導(dǎo)炎癥的細(xì)胞因子,在2008年發(fā)表在Nature雜志上的報道發(fā)現(xiàn)MIF也是代謝的調(diào)節(jié)因子,在心臟缺血狀態(tài)下可激活A(yù)MPK (AMP-activated protein kniase)通路,增加心肌對糖分的攝取,使心臟有足夠的能量供應(yīng),最終起到保護(hù)心肌的作用。而MIF基因也存在突變,MIF基因在啟動子上的一種突變可降低MIF的表達(dá),缺血心臟AMPK的激活隨之明顯減低。MIF在維持心肌穩(wěn)態(tài)中起著十分重要的作用。其次,外界刺激、疾病及年齡等后天因素對心臟功能有重要影響。本文將涉及兩個病理模型,一是內(nèi)質(zhì)網(wǎng)應(yīng)激模型,二是衰老模型,討論后天條件對心臟的影響。現(xiàn)今的環(huán)境污染、不健康的生活方式及壓力等均會導(dǎo)致內(nèi)質(zhì)網(wǎng)應(yīng)激的發(fā)生。內(nèi)質(zhì)網(wǎng)應(yīng)激在多種疾病如肥胖、糖尿病、心衰、心肌缺血再灌注中扮演重要角色。內(nèi)質(zhì)網(wǎng)是細(xì)胞內(nèi)的雙層膜結(jié)構(gòu),它的主要功能是加工新合成的蛋白,使其正確折疊,并將折疊加工好的蛋白轉(zhuǎn)運到高爾基體。同時內(nèi)質(zhì)網(wǎng)也是重要的儲存鈣離子的細(xì)胞器。正常狀態(tài)下約有30%的蛋白非正確折疊,這些蛋白將隨即被內(nèi)質(zhì)網(wǎng)中的相關(guān)機(jī)制降解。但內(nèi)質(zhì)網(wǎng)加工蛋白的過程很容易受影響,尤其當(dāng)細(xì)胞面臨缺氧、炎癥、氧化應(yīng)激等外界刺激時,使內(nèi)質(zhì)網(wǎng)處于應(yīng)激狀態(tài),激活未折疊蛋白反應(yīng)(Unfolded protein response, UPR),啟動細(xì)胞保護(hù)程序,維持內(nèi)質(zhì)網(wǎng)的穩(wěn)態(tài)。但若外界刺激持續(xù)存在,可導(dǎo)致UPR過度激活,發(fā)生內(nèi)質(zhì)網(wǎng)應(yīng)激(Endoplasmic reticulum stress, ER stress)。衣霉素(Tunicamycin)可導(dǎo)致細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激,被廣泛應(yīng)用于內(nèi)質(zhì)網(wǎng)應(yīng)激模型的建立。衰老一直是人類難以克服的難題。衰老伴隨著器官功能退化,細(xì)胞代謝水平降、低,細(xì)胞器功能減低以及相關(guān)分子表達(dá)水平和應(yīng)答能力的改變。心臟衰老表現(xiàn)出明顯的心臟收縮及舒張功能的減低、心肌能量代謝的下降以及處理鈣離子能力的降低。目前的研究表明,衰老導(dǎo)致的心臟功能變化的分子機(jī)制包括氧化應(yīng)激、線粒體功能下降等,但更詳細(xì)的機(jī)制仍有待人類的探索。自噬(Autophagy)是細(xì)胞通過自我降解衰老的或損壞的蛋白及細(xì)胞器,完成細(xì)胞自我更新及物質(zhì)重復(fù)利用的過程。細(xì)胞在正常狀態(tài)下維持一定的自噬水平,以維持細(xì)胞活力。自噬水平過低與過高都會影響心肌穩(wěn)態(tài)。自噬水平過低,心肌細(xì)胞無法完成自我更新,大量的廢物堆積,代謝減慢,進(jìn)而心肌收縮功能減低甚至死亡。自噬水平過高,心肌細(xì)胞正常的蛋白與細(xì)胞器被降解,心肌功能同樣減低甚至死亡。自噬與多種心臟疾病息息相關(guān),如心臟缺血再灌注、慢性心力衰竭、冠心病以及敗血癥引起的心衰等。mTOR (Mammalian target of rapamycin),是一種絲氨酸／蘇氨酸激酶,在生物體代謝與生長過程中起重要調(diào)節(jié)作用。mTOR同時也是最重要的自噬負(fù)性調(diào)節(jié)蛋白,在自噬經(jīng)典通路中起重要作用。mTOR被磷酸化而激活,磷酸化的mTOR可直接抑制自噬。mTOR受多種因子的調(diào)節(jié),如Akt可促進(jìn)mTOR的磷酸化,而AMPK可抑制mTOR的磷酸化。本研究將探討心肌在不同的ADH或MIF基因型中,對外界刺激—內(nèi)質(zhì)網(wǎng)應(yīng)激或衰老的應(yīng)答及其內(nèi)在機(jī)制。研究目的本文通過兩部分研究(1)內(nèi)質(zhì)網(wǎng)應(yīng)激對野生型和心臟特異性ADH過表達(dá)小鼠心臟結(jié)構(gòu)及功能的影響,以及mTOR依賴性自噬在此過程中的作用及機(jī)制； (2)衰老對野生型和MIF敲基因小鼠心臟結(jié)構(gòu)和功能的影響,以及mTOR依賴性自噬在此過程中的作用及機(jī)制,探討外界刺激或病理狀態(tài)(內(nèi)質(zhì)網(wǎng)應(yīng)激、衰老)對心臟結(jié)構(gòu)與功能的影響,以及ADH與MIF蛋白在此過程中的作用及內(nèi)在機(jī)制。機(jī)制研究著重探討mTOR依賴性自噬是否發(fā)揮作用及相關(guān)的上游調(diào)節(jié)機(jī)制。研究方法第一部分乙醇脫氫酶通過減輕氧化應(yīng)激及自噬保護(hù)內(nèi)質(zhì)網(wǎng)應(yīng)激引起的心臟收縮功能紊亂—PTEN-Akt-mTOR通路的作用1.動物模型野生型(FVB)小鼠和ADH心臟特異性過表達(dá)小鼠分別被注射衣霉素(1mg/kg,1次)或相同體積PBS,48小時后進(jìn)行實驗。2.心臟功能與心肌收縮功能及鈣離子處理能力的檢測通過多普勒超聲研究心臟功能；通過Langendorff系統(tǒng)及Librase酶離體分離小鼠心肌細(xì)胞,在SoftEdge Myocam軟件中記錄心肌細(xì)胞的收縮功能,并應(yīng)用fura-2/AM熒光在雙向激發(fā)熒光相位倍增系統(tǒng)中記錄心肌細(xì)胞鈣離子攝取情況。3.氧化應(yīng)激水平的檢測應(yīng)用H2DCFDA和DHE染色法分析從小鼠心臟分離的心肌細(xì)胞的氧化應(yīng)激水平。4.蛋白表達(dá)水平的檢測應(yīng)用Western Blot分析內(nèi)質(zhì)網(wǎng)應(yīng)激、自噬以及PTEN-Akt-mTOR通路相關(guān)蛋白的表達(dá)水平。5.體外實驗對分離的FVB或ADH小鼠心肌細(xì)胞,應(yīng)用衣霉素或(和)自噬抑制劑3-MA、Akt抑制劑AKTI及mTOR抑制劑rapamycin處理,在SoftEdge Myocam軟件中記錄心肌細(xì)胞的收縮功能。第二部分巨噬細(xì)胞遷移抑制因子缺陷改善衰老導(dǎo)致的心臟炎癥,但惡化衰老導(dǎo)致的心臟重構(gòu)和功能紊亂—自噬的作用1.實驗?zāi)Ｐ?1)衰老動物模型。野生型(C57BL/6)小鼠和MIF敲除小鼠分別被正常喂養(yǎng)至3-4個月,為年輕組；正常喂養(yǎng)至24個月為衰老組。(2)注射自噬激動劑Rapamycin的動物模型。腹腔注射Rapamycin (2mg/kg/d,8周)。(3)早衰模型及MIF低表達(dá)模型。應(yīng)用Doxorubicin (0.1μuM,24小時)處理H9C2細(xì)胞建立早衰模型；用MIF siRNA轉(zhuǎn)染細(xì)胞建立MIF低表達(dá)模型。2.心臟功能與心肌收縮功能及鈣離子處理能力的檢測通過多普勒超聲研究心臟功能；通過Langendorff系統(tǒng)及Librase酶離體分離小鼠心肌細(xì)胞,在SoftEdge Myocam軟件中記錄心肌細(xì)胞的收縮功能,并應(yīng)用fura-2/AM熒光在雙向激發(fā)熒光相位倍增系統(tǒng)中記錄心肌細(xì)胞鈣離子攝取情況。3.心肌組織結(jié)構(gòu)與心臟纖維化的檢測Lectin免疫染色研究心臟組織結(jié)構(gòu)；Masson Trichorme染色研究心肌纖維化。4.小鼠糖代謝與心肌ATP水平的檢測IPGTT實驗測定葡萄糖代謝,分析小鼠的葡萄糖利用能力。應(yīng)用氯仿-甲醇法用分光光度計(激發(fā)光波長350nm,發(fā)射光波長485nm)測心臟組織勻漿的ATP水平。5.蛋白表達(dá)水平的檢測應(yīng)用Western Blot分析衰老、自噬、AMPK-mTOR通路及炎癥相關(guān)蛋白的表達(dá)水平。研究結(jié)果第一部分乙醇脫氫酶通過減輕氧化應(yīng)激及自噬保護(hù)內(nèi)質(zhì)網(wǎng)應(yīng)激引起的心臟收縮功能紊亂—PTEN-Akt-mTOR通路的作用1.ADH緩解了內(nèi)質(zhì)網(wǎng)應(yīng)激引起的心臟功能下降。在衣霉素作用下,野生型小鼠的心臟功能明顯下降,而ADH過表達(dá)小鼠的心臟功能明顯優(yōu)于野生型小鼠。給予衣霉素注射后,野生型小鼠心肌攝取鈣離子的能力明顯下降,而ADH過表達(dá)小鼠鈣離子處理能力得到改善。2.ADH降低了內(nèi)質(zhì)網(wǎng)應(yīng)激導(dǎo)致的氧化應(yīng)激。3.ADH緩解了衣霉素導(dǎo)致的內(nèi)質(zhì)網(wǎng)應(yīng)激及自噬水平的升高,改善了PTEN-Akt-mTOR通路蛋白表達(dá)的下降。衣霉素的應(yīng)用導(dǎo)致野生型小鼠內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白BIP和GADD153及自噬相關(guān)蛋白LC3BI、Atg5、Atg7、P62的表達(dá)上升,而ADH過表達(dá)小鼠中,這些蛋白的上升程度均明顯低于野生型小鼠。衣霉素的應(yīng)用在野生型小鼠中同樣導(dǎo)致了PTEN、Akt、mTOR磷酸化水平的下降,而在ADH過表達(dá)小鼠中,這些分子通路蛋白的下降同樣得到了改善。4.ADH通過Akt-mTOR通路抑制自噬,保護(hù)內(nèi)質(zhì)網(wǎng)應(yīng)激引起的心肌功能紊亂。在體外研究中,衣霉素的應(yīng)用使離體心肌細(xì)胞收縮功能明顯下降,而在ADH過表心肌細(xì)胞中,或給予自噬抑制劑3-MA后,衣霉素導(dǎo)致的細(xì)胞收縮功能減低明顯得到緩解。但在ADH過表達(dá)心肌中加入Akt抑制劑AktI或mTOR抑制劑Rapamycin,ADH的抗衣霉素作用不復(fù)存在。第二部分巨噬細(xì)胞遷移抑制因子缺陷改善衰老導(dǎo)致的心臟炎癥,但惡化衰老導(dǎo)致的心臟重構(gòu)和功能紊亂—自噬的作用1.MIF缺陷惡化了衰老導(dǎo)致的糖利用功能減低及心臟功能下降。IPGTT實驗中,在給小鼠注射葡萄糖后的15分鐘和60分鐘時間點, 衰老小鼠的血糖水平明顯低于年輕小鼠,而衰老的MIF敲除小鼠降低更為明顯。衰老小鼠的心臟功能與心肌收縮功能明顯減低,而在MIF敲除小鼠中心臟功能進(jìn)一步降低。2.MIF缺陷進(jìn)一步惡化了衰老導(dǎo)致的心肌肥大與纖維化。3.衰老心肌AMPK-mTOR通路受抑制,自噬水平下降,這在MIF缺陷的衰老小鼠中更加明顯。衰老心肌的自噬標(biāo)志蛋白LC2BII表達(dá)下降,P62上升,表明自噬水平下降,廢物堆積增加；AMPK磷酸化水平的下降和mTOR磷酸化水平的增高表明AMPK-mTOR通路收到抑制。MIF缺陷進(jìn)一步加重了這些變化。4. Doxorubicin導(dǎo)致H9C2細(xì)胞早衰標(biāo)志物SA-β-Gal的表達(dá)增加,應(yīng)用MIFsiRNA加重了早衰水平,恢復(fù)MIF表達(dá)后早衰水平得到緩解。5.自噬激動劑Rapamycin緩解了MIF缺陷對衰老心臟功能的惡化。6.MIF缺陷緩解了衰老導(dǎo)致的炎癥水平升高,但通過Rapamycin激動自噬對炎癥并無明顯影響。研究結(jié)論我們的研究首次表明ADH自身對內(nèi)質(zhì)網(wǎng)應(yīng)激導(dǎo)致心功能損傷的保護(hù)作用,并進(jìn)一步說明其機(jī)制是通過緩解內(nèi)質(zhì)網(wǎng)應(yīng)激導(dǎo)致的氧化應(yīng)激水平,以及降低PTEN-Akt-mTOR通路介導(dǎo)的自噬水平起到心肌保護(hù)作用。我們的研究同樣首次表明了MIF缺陷進(jìn)一步惡化衰老導(dǎo)致的心臟功能下降。衰老導(dǎo)致自噬水平的降低,從而使心臟能量代謝及廢物更新受阻,心臟功能下降。MIF介導(dǎo)的AMPK-mTOR通路可恢復(fù)自噬水平。因此MIF的缺失導(dǎo)致自噬水平進(jìn)一步降低,心臟功能也進(jìn)一步惡化。在這兩個研究中,mTOR依賴性自噬起到了相反的作用。在ADH-內(nèi)質(zhì)網(wǎng)應(yīng)激模型中,內(nèi)質(zhì)網(wǎng)應(yīng)激導(dǎo)致了mTOR依賴性自噬的過度激活,從而細(xì)胞死亡增加,心臟功能受損；而在MIF-衰老模型中,衰老導(dǎo)致mTOR依賴性自噬的下降,使心肌代謝及供能下降,心臟功能受損。因此,為達(dá)到心肌保護(hù)的目的,應(yīng)當(dāng)使自噬維持在適當(dāng)?shù)乃健TOR是調(diào)節(jié)自噬的重要靶點,針對mTOR調(diào)節(jié)的藥物有很大的潛在臨床應(yīng)用價值。
[Abstract]:Normal cardiac function is the basic condition for ensuring human health and quality of life. However, myocardial cells belong to permanent cells and are almost nonrenewable after the differentiation is completed. Therefore, it is important to maintain normal cardiac function. However, the maintenance of cardiac function is facing many challenges. Genetic abnormalities that directly encode cardiac development related proteins can lead to cardiac abnormalities and severe cardiac dysfunction. But in healthy people, people who are of the same age, sex, body weight and height are still different in heart function, especially in the face of a certain stress challenge. This is particularly evident. This is due to human genetic polymorphisms. This article will discuss the effects of Alcohol dehydrogenase (ADH) and macrophage migration inhibitory factor (Macrophage migration inhibitory factor, MIF) on the cardiac function of the transgenic or gene knockout model..ADH will convert alcohol into acetaldehyde and complete alcohol. .ADH1, the first step in sperm metabolism, plays the most important role in alcohol metabolism as a subtype of ADH. The gene polymorphisms of human encoded ADH1 substructure ADH1B are gene polymorphisms. Mutant ADH1B carriers show super strong ADH activity. In East Asia, the mutation rate of ADH1B is 40%-90%, far higher than that in African and European populations. The ADH overexpression model is often used to study the effect of alcohol metabolism on cardiac function, but the effect of ADH protein itself on cardiac function is only less and still controversial. The study of the effect of ADH polymorphism on the heart function for East Asian population.MIF is the cytokine that mediates inflammation in T cells, published in Natu in 2008. The report in the re magazine found that MIF is also a regulatory factor in metabolism, which activates the AMPK (AMP-activated protein kniase) pathway in the ischemic state of the heart, increases the uptake of sugar in the myocardium, makes the heart sufficient energy supply, and ultimately protects the myocardium. The MIF gene also has a mutation and a mutation of the MIF gene in the promoter. It can reduce the expression of MIF, and the activation of AMPK in the ischemic heart significantly reduces the role of.MIF in the maintenance of myocardial homeostasis. Secondly, external stimuli, disease and age have important effects on cardiac function. This article will involve two pathological models, one is the endoplasmic reticulum stress model, the two is the aging model, and the postnatal days are discussed. The effects on the heart. Current environmental pollution, unhealthy lifestyles and stress can lead to endoplasmic reticulum stress. Endoplasmic reticulum stress plays an important role in a variety of diseases such as obesity, diabetes, heart failure, and myocardial ischemia reperfusion. The endoplasmic reticulum is a double layer membrane structure within the cell and its main function is to process newly synthesized eggs. White, folding it correctly, and transferring the processed protein to the Golgi apparatus. And the endoplasmic reticulum is also an important organelle for storing calcium ions. About 30% of the proteins are not properly folded in normal state, and these proteins will be degraded in the endoplasmic reticulum, but the process of protein processing in the endoplasmic reticulum is easily affected. When the cells are exposed to external stimuli such as hypoxia, inflammation, and oxidative stress, the endoplasmic reticulum is in a stress state, activating the unfolded protein reaction (Unfolded protein response, UPR), initiating the cell protection program and maintaining the homeostasis homeostasis. But if the external stimulus persists, it can lead to excessive activation of UPR and the occurrence of endoplasmic reticulum stress (Endoplasmic reticulu). M stress, ER stress). Ycomycin (Tunicamycin) can lead to endoplasmic reticulum stress and is widely used in the establishment of endoplasmic reticulum stress model. Senescence has been a difficult problem to be overcome by human beings. Aging is accompanied by degradation of organ function, decrease of cell metabolism level, low cell power, and modification of related molecular expression level and response ability. Cardiac senescence shows a significant reduction in cardiac contractile and diastolic function, a decline in cardiac energy metabolism and a decrease in the ability to deal with calcium ions. Current research shows that molecular mechanisms of cardiac function changes, including oxidative stress and decrease in mitochondrial function, are still needed to be explored by human beings. Autophagy is the process of cell self regeneration and reutilization of cells by self degradation of aging or damaged proteins and organelles. Cells maintain a certain level of autophagy to maintain cell viability under normal conditions. The low and high level of autophagy will affect the homeostasis of the myocardium. The level of autophagy is too low and the cardiac myocytes are not. Complete self renewal, accumulation of large amounts of waste, slow metabolism, and reduced myocardial contractile function or even death. High autophagy, normal protein and organelles of cardiac myocytes are degraded, cardiac function is also reduced or even death. Autophagy is closely related to a variety of heart diseases, such as cardiac ischemia reperfusion, chronic heart failure, coronary heart disease .mTOR (Mammalian target of rapamycin), which is caused by septicemia, is a serine / threonine kinase, which plays an important regulatory role in the metabolism and growth of organisms..mTOR is also the most important autophagic negative regulatory protein. It plays an important role in the classical pathway of autophagy, which is activated by phosphorylation and phosphorylation of mTOR. Autophagic.MTOR can be directly inhibited by a variety of factors, such as Akt can promote the phosphorylation of mTOR, and AMPK can inhibit the phosphorylation of mTOR. This study will explore the response of myocardium to the response to external stimulation of endoplasmic reticulum stress or senescence in different ADH or MIF genotypes and its intrinsic mechanism. The purpose of this study was to study the endoplasmic reticulum in two parts (1) endoplasmic reticulum should be studied. The effects and mechanisms of mTOR dependent autophagy on the heart structure and function of the wild type and heart specific ADH overexpressed mice; (2) the effect of senescence on the heart structure and function of wild type and MIF knockout mice, as well as the role and mechanism of mTOR dependent autophagy in this process, and to explore the external stimuli or the external stimuli. The effect of the pathological state (endoplasmic reticulum stress, aging) on the structure and function of the heart, and the role of ADH and MIF protein in this process and its intrinsic mechanism. The mechanism study focuses on the role of mTOR dependent autophagy and the related upstream regulation mechanism. Cardiac contractile dysfunction induced by endoplasmic reticulum stress - the effect of PTEN-Akt-mTOR pathway 1. animal model wild type (FVB) mice and ADH heart specific overexpressed mice were injected with mycophencin (1mg/kg, 1 times) or the same volume PBS respectively. After 48 hours, the test of.2. heart function and myocardial contractile function and calcium ion processing ability were examined. The cardiac function was measured by Doppler ultrasound. The systolic function of cardiac myocytes was recorded in SoftEdge Myocam software through the isolation of Langendorff system and Librase enzyme in vitro, and the fura-2/AM fluorescence was used to record the level of.3. oxidative stress in the myocardial cell calcium uptake in the bi-directional excitation fluorescence phase multiplier system. Detection and application of H2DCFDA and DHE staining methods to detect the level of oxidative stress level.4. protein expression from cardiac isolated cardiomyocytes in mice by Western Blot analysis of endoplasmic reticulum stress, autophagy and the expression level of PTEN-Akt-mTOR pathway related proteins in.5. in vitro experiments on isolated FVB or ADH murine cardiomyocytes, using mycophenycin Or (and) (and) autophagic inhibitor 3-MA, Akt inhibitor AKTI and mTOR inhibitor rapamycin treatment, recording cardiac contractile function in SoftEdge Myocam software. Second partial macrophage migration inhibitory factor defect improves aging induced heart inflammation, but deteriorates senescence caused by cardiac remodeling and dysfunction - the role of autophagy in the 1. experimental model Type (1) aging animal model. Wild type (C57BL/6) mice and MIF knockout mice were fed to 3-4 months, respectively, for young group; normal feeding to 24 months as senescence group. (2) animal model of autophagic agonist Rapamycin injection. Intraperitoneal injection of Rapamycin (2mg/kg/d, 8 weeks). (3) premature failure model and MIF low expression model. Doxorubicin (0.1). UM, 24 hours) treatment of H9C2 cells to establish premature aging model; MIF siRNA transfected cells to establish MIF low expression model.2. cardiac function and myocardial contractile function and calcium ion processing ability detection by Doppler ultrasound study of cardiac function; Langendorff system and Librase enzyme isolation of mouse cardiomyocytes in SoftEdge Myocam soft The systolic function of cardiac myocytes was recorded in the part, and fura-2/AM fluorescence was used to record cardiac tissue structure of myocardial cells and cardiac fibrosis in the bi-directional excitation fluorescence phase multiplier system. The structure of cardiac tissue and cardiac fibrosis were detected by Lectin immunostaining. Masson Trichorme staining was used to study the glucose metabolism in.4. mice of myocardial fibrosis. Glucose metabolism was measured by IPGTT test with myocardial ATP level, and the glucose utilization ability of mice was analyzed. The detection of ATP level of ATP level of cardiac tissue homogenate by chloroform methanol spectrophotometer (excitation light wavelength 350nm, emission wavelength 485nm) should be detected by Western Blot to analyze senescence, autophagy, AMPK-mTOR pathway and The expression level of inflammation related proteins. Part 1 of the study, ethanol dehydrogenase alleviated the decline in cardiac function induced by endoplasmic reticulum stress by reducing oxidative stress and protecting endoplasmic reticulum stress dysfunction caused by endoplasmic reticulum stress and the effect of PTEN-Akt-mTOR pathway on cardiac dysfunction induced by endoplasmic reticulum stress. In the action of ycomycin, heart work of wild type mice The heart function of ADH overexpressed mice was obviously better than that of wild type mice. After injection of ycomycin, the ability of calcium ion uptake in the myocardium of wild type mice decreased significantly, while the ability of calcium ion treatment in ADH overexpressed mice was improved by.2.ADH and the oxidative stress caused by endoplasmic reticulum stress was reduced by.3.ADH. The increase of endoplasmic reticulum stress and autophagy increased the decrease in the expression of PTEN-Akt-mTOR pathway protein. The application of mycophencin led to the increase in the expression of BIP and GADD153 and autophagy related protein LC3BI, Atg5, Atg7 and P62 in the endoplasmic reticulum of wild type mice, and the increase of these proteins in ADH overexpressed mice was significantly lower than that in the wild mice. The application of ycomycin in the wild type mice also led to a decrease in the phosphorylation level of PTEN, Akt, and mTOR in the wild mice, and in the ADH overexpressed mice, the decrease of these molecular pathway proteins also improved the inhibition of autophagy through the Akt-mTOR pathway and the protection of the myocardial dysfunction induced by the endoplasmic reticulum. In vitro studies, in vitro The application of mycophentin reduced the contractile function of isolated cardiomyocytes, and the decrease of contractile function caused by mycophenin was obviously alleviated in the ADH overexpressed cardiomyocytes or after the autophagy inhibitor 3-MA was given, but the effect of the Akt inhibitor AktI or mTOR inhibitor Rapamycin in the ADH overexpressed myocardium was no longer in existence. The second part of the macrophage migration inhibitory factor deficiency improves the heart inflammation caused by aging, but the cardiac remodeling and dysfunction caused by the deterioration of aging - the role of autophagy 1.MIF deficiency worsens the aging induced impaired glucose utilization and the decline of the heart function in the.IPGTT experiment, when the glucose injection is given to mice for 15 and 60 minutes The blood sugar level of aging mice was significantly lower than that of young mice, while the aging MIF knockout mice decreased more obviously. The cardiac function and myocardial contractile function of aging mice decreased significantly, while in MIF knockout mice, the heart function further reduced the.2.MIF deficiency and further worsened the myocardial hypertrophy induced by aging and the fibrosis of the.3. senescence heart. The AMPK-mTOR pathway was inhibited and autophagy decreased, which was more obvious in the aging mice with MIF defects. The expression of autophagic marker protein LC2BII in the aging myocardium decreased, P62 increased, indicating that the level of autophagy decreased and the accumulation of waste increased; the decline of AMPK phosphorylation level and the increase of mTOR phosphorylation level indicated that the AMPK-mTOR pathway received the inhibition of.MIF. Defects further aggravate these changes..4. Doxorubicin leads to increased expression of SA- beta -Gal in H9C2 cell premature senescence, MIFsiRNA plus
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R541.6
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本文編號：2044187