【摘要】：目的：在機(jī)體的適應(yīng)性免疫和宿主的防御系統(tǒng)中，T細(xì)胞的抗原依賴性激活扮演著十分重要的角色。而在此過(guò)程中，最為關(guān)鍵的一步便是II型主要組織相容性復(fù)合物（MHC II）的表達(dá)激活。CIITA作為MHC II的反式激活因子，能夠上調(diào)MHCII的表達(dá)，因此關(guān)于CIITA的活性調(diào)控近年來(lái)成為了人們研究的重點(diǎn)。我們主要是從翻譯后修飾水平來(lái)探討去乙�；瘜�(duì)于CIITA活性的影響。 SIRT1作為NAD+依賴的組蛋白去乙�；�，它能夠通過(guò)調(diào)節(jié)組蛋白或者相關(guān)轉(zhuǎn)錄因子的乙�；絽⑴c到眾多的生物學(xué)過(guò)程中去。我們之前的研究已經(jīng)發(fā)現(xiàn)一種I型去乙�；福ńM蛋白去乙�；�2/HDAC2）能夠與CIITA結(jié)合并去乙�；疌IITA，使其蛋白穩(wěn)定性下降，從而抑制其活性。而近期的一些研究表明，III型組蛋白去乙�；窼IRT1在調(diào)控免疫系統(tǒng)過(guò)程中也發(fā)揮著一定作用。鑒于MHC II的轉(zhuǎn)錄激活在免疫系統(tǒng)中的關(guān)鍵地位，我們?cè)噲D去探求SIRT1對(duì)于該激活過(guò)程有何影響。 方法：我們分別在人胚胎腎細(xì)胞（293）,人單核巨噬細(xì)胞（THP-1）以及小鼠的巨噬細(xì)胞（RAW264.7）中，通過(guò)實(shí)時(shí)定量PCR和熒光素酶報(bào)告基因活性分析實(shí)驗(yàn)檢測(cè)SIRT1對(duì)于MHC II的mRNA和轉(zhuǎn)錄水平的影響。通過(guò)免疫共沉淀和Western blotting檢測(cè)SIRT1與CIITA之間的結(jié)合以及CIITA乙�；阶兓�。通過(guò)半衰期實(shí)驗(yàn)檢測(cè)SIRT1對(duì)于CIITA蛋白穩(wěn)定性的作用，并利用ChIP實(shí)驗(yàn)檢測(cè)SIRT1對(duì)于CIITA結(jié)合到MHC II啟動(dòng)子上的影響。最后在缺氧和ox-LDL兩種應(yīng)激條件下，我們用IP實(shí)驗(yàn)檢測(cè)了CIITA乙�；降淖兓�，并用染色質(zhì)免疫共沉淀（ChIP）和報(bào)告基因?qū)嶒?yàn)進(jìn)一步研究在這兩種條件下CIITA對(duì)于MHCII的轉(zhuǎn)錄調(diào)控水平的變化。 結(jié)果：在巨噬細(xì)胞中，SIRT1能夠上調(diào)CIITA對(duì)于MHC II的轉(zhuǎn)錄激活作用，其具體機(jī)制是，SIRT1能夠與CIITA結(jié)合，通過(guò)發(fā)揮其去乙�；傅淖饔�，一方面能夠增強(qiáng)CIITA的蛋白穩(wěn)定性，使其半衰期延長(zhǎng)；另一方面能夠促進(jìn)促進(jìn)CIITA的核積聚并結(jié)合到MHC II啟動(dòng)子上，從而激活其轉(zhuǎn)錄表達(dá)。 在缺氧和oxLDL處理情況下，由于SIRT1的轉(zhuǎn)錄表達(dá)水平和活性均有所下降，，CIITA的轉(zhuǎn)錄激活作用受到抑制。而當(dāng)給與白藜蘆醇處理之后，這種抑制作用有所緩解。 結(jié)論：作為NAD+依賴性的去乙�；�，SIRT1能夠與CIITA結(jié)合，通過(guò)組蛋白修飾途徑，一方面增加CIITA的蛋白穩(wěn)定性,另一方面促使其核集聚增加其結(jié)合到MHC II啟動(dòng)子水平，從而上調(diào)MHC II的表達(dá)。由此，我們將SIRT1調(diào)節(jié)的CIITA的去乙�；途奘杉�(xì)胞中MHC II的激活聯(lián)系在一起，這也讓我們對(duì)于適應(yīng)性免疫系統(tǒng)在一些異常刺激之下的反應(yīng)有了更深的理解。 目的：心血管疾病是威脅人類健康的最常見(jiàn)疾病。目前我國(guó)正處于心血管疾病爆發(fā)的“窗口期”。根據(jù)我國(guó)流行病學(xué)調(diào)查，由于人們生活方式以及飲食習(xí)慣的變化，其發(fā)病率和死亡率均呈現(xiàn)不斷上升的趨勢(shì)。作為心血管疾病中的一個(gè)重要分支，心肌梗死是由冠狀動(dòng)脈粥樣硬化引起血栓形成、冠狀動(dòng)脈的分支堵塞，使一部分心肌失去血液供應(yīng)而壞死的病癥。隨著醫(yī)學(xué)發(fā)展和科技進(jìn)步，心血管疾病的基礎(chǔ)研究，取得了很大的進(jìn)展，對(duì)疾病的認(rèn)識(shí)，已從整體和組織水平，不斷向細(xì)胞和分子水平深入。 SIRT1作為NAD+依賴的組蛋白去乙�；�，它能夠通過(guò)調(diào)節(jié)組蛋白或者相關(guān)轉(zhuǎn)錄因子的乙�；絽⑴c到眾多的生物學(xué)過(guò)程中去，其中關(guān)于SIRT1對(duì)于心肌以及心臟功能保護(hù)作用的研究報(bào)道已經(jīng)屢見(jiàn)不鮮。鑒于SIRT1重要的病理生理學(xué)意義，從轉(zhuǎn)錄水平去研究SIRT1活性調(diào)控成為了人們研究的重點(diǎn)，在它的啟動(dòng)子區(qū)域已經(jīng)發(fā)現(xiàn)了許多轉(zhuǎn)錄因子結(jié)合位點(diǎn)，正是在這些轉(zhuǎn)錄因子復(fù)雜的調(diào)控機(jī)制之下，SIRT1的轉(zhuǎn)錄活性維持在一個(gè)相對(duì)穩(wěn)定的水平。其中，關(guān)于組蛋白甲基化對(duì)于SIRT1的轉(zhuǎn)錄調(diào)控機(jī)制方面的研究幾乎沒(méi)有什么報(bào)道，因此我們想看一下組蛋白甲基化對(duì)于SIRT1的轉(zhuǎn)錄水平是否存在一定影響，而這一機(jī)制對(duì)于心肌缺血所導(dǎo)致的心梗模型又有怎樣的作用，這些問(wèn)題都是我們需要探討的。 方法：我們分別在人胚胎腎細(xì)胞（293）以及大鼠的心肌細(xì)胞（H9C2）中給予MTA（廣譜的甲基化轉(zhuǎn)移酶抑制劑）處理，通過(guò)實(shí)時(shí)定量PCR和熒光素酶報(bào)告基因活性分析實(shí)驗(yàn)檢測(cè)MTA對(duì)于SIRT1的mRNA和轉(zhuǎn)錄水平的影響。通過(guò)Western blotting檢測(cè)MTA對(duì)于SIRT1蛋白水平影響。利用報(bào)告基因?qū)嶒?yàn)我們篩選出潛在的甲基化轉(zhuǎn)移酶SUV39h。為了進(jìn)一步驗(yàn)證其特異性，我們購(gòu)買了SUV39H的特異性抑制劑chaetocin。當(dāng)上述細(xì)胞給與該抑制劑處理后，同樣利用報(bào)告基因、RT-PCR以及Western blotting檢測(cè)了SIRT1轉(zhuǎn)錄表達(dá)水平。在H9C2細(xì)胞中，利用siRNA將SUV39h1和SUV39h2敲除之后檢測(cè)SIRT1的轉(zhuǎn)錄水平。最后，我們通過(guò)體內(nèi)和體外實(shí)驗(yàn)構(gòu)建大鼠心肌梗死模型來(lái)檢測(cè)該藥物對(duì)于心梗是否具有一定的保護(hù)作用。 結(jié)果：在心肌細(xì)胞中，無(wú)論是給予MTA還是Chaetocin處理,SIRT1的轉(zhuǎn)錄水平以及表達(dá)水平均有所上升，通過(guò)篩選我們發(fā)現(xiàn)它們作用靶點(diǎn)可能是組蛋白甲基化轉(zhuǎn)移酶SUV39h，該家族有2個(gè)成員。利用siRNA技術(shù)，我們發(fā)現(xiàn)只有SUV39h2發(fā)揮作用。在體內(nèi)和體外模型中，我們發(fā)現(xiàn)給予Chaetocin處理的確能夠緩解心肌缺血所造成的心肌梗死。我們的研究將組蛋白的甲基化水平與心肌梗死癥狀聯(lián)系在一起，進(jìn)一步證明了SIRT1在保護(hù)心臟功能方面的重要作用。
[Abstract]:AIM: Antigen-dependent activation of T cells plays an important role in the adaptive immune system and host defense system. The key step in this process is the activation of expression of major histocompatibility complex type II (MHC II). Therefore, the regulation of CIITA activity has become the focus of research in recent years. We mainly discuss the effect of deacetylation on CIITA activity from the level of post-translational modification.
SIRT1, a NAD+ dependent histone deacetylase, can participate in many biological processes by regulating acetylation levels of histones or related transcription factors. Our previous studies have found that a type I deacetylase (histone deacetylase 2/HDAC2) binds to CIITA and deacetylates it. Recent studies have shown that SIRT1, a histone deacetylase type III, also plays a role in regulating the immune system. In view of the critical role of MHC II transcriptional activation in the immune system, we attempt to explore how SIRT1 affects this activation process.
METHODS: The effects of SIRT1 on the mRNA and transcriptional levels of MHC II in human embryonic kidney cells (293), human monocyte-macrophages (THP-1) and mouse macrophages (RAW264.7) were detected by real-time quantitative PCR and luciferase reporter gene activity assay. The effect of SIRT1 on the stability of CIITA protein was examined by half-life assay, and the effect of SIRT1 on the binding of CIITA to MHC II promoter was detected by ChIP assay. Finally, under hypoxia and ox-LDL stress, the changes of CIITA acetylation level were detected by IP assay. Chromatin immunoprecipitation (ChIP) and reporter gene assay were used to investigate the changes of transcriptional regulation of MHCII by CIITA under these two conditions.
RESULTS: SIRT1 could up-regulate the transcriptional activation of CIITA to MHC II in macrophages. The specific mechanism was that SIRT1 could bind to CIITA and enhance the protein stability of CIITA and prolong its half-life by exerting its deacetylase activity. On the other hand, SIRT1 could promote the nuclear accumulation of CIITA and its binding to MH. The C II promoter activates its transcriptional expression.
Under hypoxia and oxLDL treatment, the transcriptional activation of CIITA was inhibited due to the decrease of SIRT1 transcriptional expression and activity, which was alleviated by resveratrol treatment.
CONCLUSION: As a NAD+dependent deacetylase, SIRT1 can bind to CIITA through histone modification pathway, on the one hand, increase the protein stability of CIITA, on the other hand, promote its nuclear agglomeration to increase its binding to MHC II promoter level, thereby up-regulating the expression of MHC II. The activation of MHC II in cells is linked to a deeper understanding of the adaptive immune system's response to abnormal stimuli.
Objective:Cardiovascular disease is the most common disease threatening human health.At present,China is in the "window period" of cardiovascular disease outbreak.According to the epidemiological investigation in our country,the morbidity and mortality of cardiovascular disease are on the rise because of the changes of people's lifestyle and dietary habits. Branch, myocardial infarction is caused by coronary atherosclerosis thrombosis, coronary artery branch blockage, so that part of the myocardial blood supply loss and necrosis of the disease.With the development of medicine and scientific and technological progress, the basic research of cardiovascular disease, has made great progress, the understanding of the disease, from the overall and organizational level, has been constantly. Deeper into cellular and molecular level.
SIRT1, as a NAD+ dependent histone deacetylase, can participate in many biological processes by regulating the acetylation level of histones or related transcription factors. Among them, there are many reports about the protective effects of SIRT1 on myocardium and cardiac function. The study of SIRT1 activity regulation at transcriptional level has become the focus of research. Many transcription factor binding sites have been found in its promoter region. It is precisely under the complex regulatory mechanisms of these transcription factors that the transcriptional activity of SIRT1 is maintained at a relatively stable level. So we want to see if histone methylation has some effect on the transcriptional level of SIRT1, and what effect this mechanism has on myocardial infarction model induced by myocardial ischemia, which we need to explore.
METHODS: MTA (broad-spectrum methyltransferase inhibitor) was administered to human embryonic kidney cells (293) and rat cardiomyocytes (H9C2) respectively. The effect of MTA on SIRT1 mRNA and transcription level was detected by real-time quantitative PCR and luciferase reporter gene activity assay. The effect of MTA on SIRT1 was detected by Western blotting. To further verify the specificity, we purchased chaetocin, a specific inhibitor of SUV39H. When these cells were treated with the inhibitor, the SIRT1 transcription table was also detected by reporter gene, RT-PCR and Western blotting. In H9C2 cells, SUV39h1 and SUV39h2 were knocked out by siRNA to detect the transcriptional level of SIRT1. Finally, we constructed a rat model of myocardial infarction in vivo and in vitro to test whether the drug has a protective effect on myocardial infarction.
Results: Both MTA and Chaetocin treatment increased the transcription and expression of SIRT1 in cardiomyocytes. Through screening, we found that the target of SIRT1 might be histone methylation transferase SUV39h, which has two members. In vitro models, we found that chaetocin treatment did alleviate myocardial infarction caused by myocardial ischemia. Our study linked histone methylation levels with myocardial infarction symptoms, further demonstrating the important role of SIRT1 in protecting cardiac function.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前2條

1 曹春雨;熱應(yīng)激細(xì)胞中CIITA基因的表觀遺傳調(diào)控研究[D];北京協(xié)和醫(yī)學(xué)院;2012年

2 夏駿;肺部免疫反應(yīng)和血管重塑中重要轉(zhuǎn)錄事件調(diào)控機(jī)制的研究[D];南京醫(yī)科大學(xué);2013年

相關(guān)碩士學(xué)位論文 前1條

1 孔小岑;脫乙酰酶在巨噬細(xì)胞和平滑肌細(xì)胞中對(duì)CIITA轉(zhuǎn)錄活性的調(diào)節(jié)[D];南京醫(yī)科大學(xué);2010年

本文編號(hào)：2230432