本文選題：胰島素　切入點(diǎn)：過氧化氫　出處：《吉林大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：背景： 目前急性心肌梗死發(fā)病率逐年增加,已成為人類死亡率最高的疾病之一。心肌梗死過程中心肌細(xì)胞缺血缺氧會(huì)造成心肌細(xì)胞壞死和凋亡，梗死灶的中央通常以心肌壞死為主，梗死灶周邊部分以細(xì)胞凋亡為主。冠狀動(dòng)脈血運(yùn)重建治療如支架術(shù)可以迅速開通血管，避免心肌壞死的進(jìn)一步擴(kuò)大，同是也伴隨著心肌缺血再灌注損傷，此種心肌損傷也包括心肌細(xì)胞壞死和凋亡，心肌細(xì)胞的壞死和凋亡造成心肌細(xì)胞數(shù)量減少是引起心力衰竭發(fā)生發(fā)展的原因之一。如何減少心肌細(xì)胞在缺血缺氧條件下的凋亡成為現(xiàn)今心肌梗死研究的熱點(diǎn)。尋找方法阻斷誘導(dǎo)心肌細(xì)胞凋亡的信號(hào)或阻斷將這些信號(hào)與死亡程序鏈接起來的通道將有助于阻遏凋亡，防止心肌細(xì)胞數(shù)量減少，以預(yù)防或改善心功能。近期文獻(xiàn)報(bào)道m(xù)icroRNA-210在急性心肌梗死過程和缺血再灌注中具有抗凋亡的作用，有望成為治療急性心肌梗死的MicroRNA，但其具體作用和機(jī)制尚未闡明。 氧化應(yīng)激在心肌缺血再灌注心肌細(xì)胞壞死凋亡過程中具有重要作用。氧化應(yīng)激是體內(nèi)活性氧自由基產(chǎn)生過多，氧化系統(tǒng)和抗氧化系統(tǒng)失衡，從而導(dǎo)致組織和細(xì)胞的損傷。自由基是過多的活性氧和活性氮自由基產(chǎn)生的，可以損傷脂肪、蛋白質(zhì)和核苷酸�；钚匝踝杂苫⒌杂苫瓦^氧化氫在心臟心肌缺血/再灌注中損傷中發(fā)揮著重要的作用。缺血再灌注過程中可以產(chǎn)生H2O2，并且在氧化應(yīng)激損傷中是氧自由基的供體。H2O2在氧化應(yīng)激心肌損傷中基因表達(dá)、翻譯的作用機(jī)制尚未闡明，尤其是其對(duì)于microRNA的影響并不清楚。 研究表明Akt是miRNA-210的上游信號(hào)通路，胰島素是經(jīng)典的信號(hào)通路PI3K-AKT的激動(dòng)劑，同時(shí)胰島素具有抗氧化應(yīng)激作用。因此我們提出科學(xué)假設(shè)：胰島素可通過激活經(jīng)典的PI3K-AKT信號(hào)通路，導(dǎo)致miRNA-210增加，而miRNA-210具有抗凋亡作用，進(jìn)而發(fā)揮抗氧化應(yīng)激、抗凋亡作用。本研究擬通過細(xì)胞學(xué)實(shí)驗(yàn)，通過實(shí)時(shí)定量PCR、MTT、Western Blot、流式細(xì)胞儀檢測(cè)等方法驗(yàn)證本科學(xué)假設(shè)的正確性。 方法： 本研究應(yīng)用大鼠心肌細(xì)胞系H9c2細(xì)胞，進(jìn)行如下實(shí)驗(yàn)： （1）采用不同濃度的過氧化氫處理H9c2心肌細(xì)胞，，采用MTT方法和流式細(xì)胞儀檢測(cè)方法檢測(cè)心肌細(xì)胞生存率和凋亡率； （2）應(yīng)用胰島素預(yù)處理H9c2心肌細(xì)胞，然后應(yīng)用過氧化氫處理，采用MTT方法和流式細(xì)胞儀檢測(cè)方法檢測(cè)心肌細(xì)胞生存率和凋亡率； （3）采用不同濃度的過氧化氫處理H9c2心肌細(xì)胞，實(shí)時(shí)定量PCR方法檢測(cè)miRNA-210表達(dá)相對(duì)量；采用同濃度的過氧化氫處理H9c2心肌細(xì)胞不同時(shí)間，實(shí)時(shí)定量PCR方法檢測(cè)miRNA-210表達(dá)相對(duì)量； （4）應(yīng)用不同濃度的胰島素處理H9c2心肌細(xì)胞，實(shí)時(shí)定量PCR方法檢測(cè)miRNA-210表達(dá)相對(duì)量；采用同濃度的胰島素處理H9c2心肌細(xì)胞不同時(shí)間，實(shí)時(shí)定量PCR方法檢測(cè)miRNA-210表達(dá)相對(duì)量； （5）采用胰島素預(yù)處理H9c2心肌細(xì)胞，采用同濃度的過氧化氫處理H9c2心肌細(xì)胞不同的時(shí)間，然后采用實(shí)時(shí)定量PCR方法檢測(cè)miRNA-210表達(dá)相對(duì)量； （6）采用慢病毒載體構(gòu)建miRNA-210過表達(dá)和低表達(dá)H9c2細(xì)胞模型； （7）應(yīng)用過氧化氫處理miRNA-210過表達(dá)和低表達(dá)H9c2心肌細(xì)胞，流式細(xì)胞儀檢測(cè)心肌細(xì)胞凋亡率； （8）應(yīng)用胰島素處理miRNA-210低表達(dá)的H9c2心肌細(xì)胞，然后應(yīng)用過氧化氫處理，流式細(xì)胞儀檢測(cè)心肌細(xì)胞凋亡率； （9）采用Western Blot法檢測(cè)胰島素和/或過氧化氫處理心肌細(xì)胞磷酸化Akt/總Akt表達(dá)比率； （10）應(yīng)用PI3K/Akt阻斷劑LY294002預(yù)處理H9c2心肌細(xì)胞，然后應(yīng)用胰島素和/或過氧化氫處理，實(shí)時(shí)定量PCR檢測(cè)miRNA-210相對(duì)表達(dá)量。 結(jié)果： 1、過氧化氫可隨濃度增加引起H9c2細(xì)胞凋亡增加； 2、胰島素具有對(duì)抗過氧化氫所致H9c2細(xì)胞凋亡的保護(hù)作用； 3、過氧化氫、胰島素及胰島素+過氧化氫均可以引起miRNA-210表達(dá)增加； 4、miRNA-210過表達(dá)具有對(duì)抗過氧化氫致心肌凋亡保護(hù)作用；miRNA-210低表達(dá)可以降低胰島素的心肌保護(hù)作用； 5、應(yīng)用LY294002阻斷PI3K-Akt信號(hào)通路，可以使胰島素、過氧化氫及胰島素+過氧化氫引起的miRNA-210表達(dá)降低。 結(jié)論： 胰島素可通過PI3K/Akt信號(hào)通路上調(diào)miRNA-210發(fā)揮抗過氧化氫致H9c2細(xì)胞凋亡作用，本研究為胰島素抗氧化應(yīng)激的作用機(jī)制提供證據(jù)。
[Abstract]:Background:
The incidence of acute myocardial infarction increased year by year, has become one of the highest mortality rate of human diseases. The process of myocardial infarction caused by hypoxia ischemia myocytes necrosis and apoptosis of myocardial infarction, the central is generally based on myocardial necrosis, the infarct peripheral part mainly apoptosis. Coronary artery revascularization treatment such as stenting can the rapid opening of blood vessels, to avoid further expansion of myocardial necrosis, the same is also accompanied by myocardial ischemia reperfusion injury, myocardial injury that also includes myocardial cell necrosis and apoptosis, necrosis and apoptosis of myocardial cells caused by the reduction of the number of myocardial cells is one of the reasons for the occurrence and development of heart failure. How to reduce myocardial cell apoptosis in ischemic and hypoxic conditions become a hot topic of today's research method for myocardial infarction. Myocardial cell apoptosis induced by blocking the signal or the signal blocking And death program link channel will help to prevent the repression of apoptosis, decrease of myocardial cell, to prevent or improve heart function. Recently reported that microRNA-210 has anti apoptotic role in acute myocardial infarction and ischemia reperfusion in the treatment of acute myocardial infarction, is expected to become MicroRNA, but its specific mechanism has not been elucidated.
Oxidative stress plays an important role in myocardial ischemia reperfusion myocardial cell apoptosis process. Oxidative stress is the oxygen free radical in vivo activity produces too much oxidation and antioxidant system imbalance, resulting in tissue and cell injury. Free radicals are excessive reactive oxygen and active nitrogen generation of free radicals, can damage the fat, protein and nucleotide. Oxygen free radicals, nitrogen free radicals and hydrogen peroxide in myocardial ischemia / reperfusion injury in the heart plays an important role. H2O2 can produce ischemia reperfusion process, and is the donor of.H2O2 oxygen free radical gene expression in myocardial oxidative stress injury in oxidative stress injury, effect the translation mechanism has not been elucidated, especially the effect of microRNA is not clear.
Research shows that Akt is the upstream signal pathway of miRNA-210, insulin signaling pathway is a classic PI3K-AKT agonist, while insulin has anti oxidative stress. So we put forward the scientific hypothesis that insulin can activate the PI3K-AKT signaling pathway classic, lead to an increase in miRNA-210, while the miRNA-210 has anti apoptotic effects, and then play against oxidative stress, anti apoptosis. This study by cytology experiment by real-time quantitative PCR, MTT, Western Blot, the accuracy of hypothesis of flow cytometry and other methods to verify the science.
Method錛

本文編號(hào)：1641031