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1、SIRT1在CIITA介导MHC Ⅱ转录激活中的作用 2、组蛋白甲基化转移酶SUV39H2在心梗中的作用机制初探

发布时间:2018-09-08 11:50
【摘要】:目的:在机体的适应性免疫和宿主的防御系统中,T细胞的抗原依赖性激活扮演着十分重要的角色。而在此过程中,最为关键的一步便是II型主要组织相容性复合物(MHC II)的表达激活。CIITA作为MHC II的反式激活因子,能够上调MHCII的表达,因此关于CIITA的活性调控近年来成为了人们研究的重点。我们主要是从翻译后修饰水平来探讨去乙酰化对于CIITA活性的影响。 SIRT1作为NAD+依赖的组蛋白去乙酰化酶,它能够通过调节组蛋白或者相关转录因子的乙酰化水平参与到众多的生物学过程中去。我们之前的研究已经发现一种I型去乙酰化酶(组蛋白去乙酰化酶2/HDAC2)能够与CIITA结合并去乙酰化CIITA,使其蛋白稳定性下降,从而抑制其活性。而近期的一些研究表明,III型组蛋白去乙酰化酶SIRT1在调控免疫系统过程中也发挥着一定作用。鉴于MHC II的转录激活在免疫系统中的关键地位,我们试图去探求SIRT1对于该激活过程有何影响。 方法:我们分别在人胚胎肾细胞(293),人单核巨噬细胞(THP-1)以及小鼠的巨噬细胞(RAW264.7)中,通过实时定量PCR和荧光素酶报告基因活性分析实验检测SIRT1对于MHC II的mRNA和转录水平的影响。通过免疫共沉淀和Western blotting检测SIRT1与CIITA之间的结合以及CIITA乙酰化水平变化。通过半衰期实验检测SIRT1对于CIITA蛋白稳定性的作用,并利用ChIP实验检测SIRT1对于CIITA结合到MHC II启动子上的影响。最后在缺氧和ox-LDL两种应激条件下,我们用IP实验检测了CIITA乙酰化水平的变化,并用染色质免疫共沉淀(ChIP)和报告基因实验进一步研究在这两种条件下CIITA对于MHCII的转录调控水平的变化。 结果:在巨噬细胞中,SIRT1能够上调CIITA对于MHC II的转录激活作用,其具体机制是,SIRT1能够与CIITA结合,通过发挥其去乙酰化酶的作用,一方面能够增强CIITA的蛋白稳定性,使其半衰期延长;另一方面能够促进促进CIITA的核积聚并结合到MHC II启动子上,从而激活其转录表达。 在缺氧和oxLDL处理情况下,由于SIRT1的转录表达水平和活性均有所下降,,CIITA的转录激活作用受到抑制。而当给与白藜芦醇处理之后,这种抑制作用有所缓解。 结论:作为NAD+依赖性的去乙酰化酶,SIRT1能够与CIITA结合,通过组蛋白修饰途径,一方面增加CIITA的蛋白稳定性,另一方面促使其核集聚增加其结合到MHC II启动子水平,从而上调MHC II的表达。由此,我们将SIRT1调节的CIITA的去乙酰化和巨噬细胞中MHC II的激活联系在一起,这也让我们对于适应性免疫系统在一些异常刺激之下的反应有了更深的理解。 目的:心血管疾病是威胁人类健康的最常见疾病。目前我国正处于心血管疾病爆发的“窗口期”。根据我国流行病学调查,由于人们生活方式以及饮食习惯的变化,其发病率和死亡率均呈现不断上升的趋势。作为心血管疾病中的一个重要分支,心肌梗死是由冠状动脉粥样硬化引起血栓形成、冠状动脉的分支堵塞,使一部分心肌失去血液供应而坏死的病症。随着医学发展和科技进步,心血管疾病的基础研究,取得了很大的进展,对疾病的认识,已从整体和组织水平,不断向细胞和分子水平深入。 SIRT1作为NAD+依赖的组蛋白去乙酰化酶,它能够通过调节组蛋白或者相关转录因子的乙酰化水平参与到众多的生物学过程中去,其中关于SIRT1对于心肌以及心脏功能保护作用的研究报道已经屡见不鲜。鉴于SIRT1重要的病理生理学意义,从转录水平去研究SIRT1活性调控成为了人们研究的重点,在它的启动子区域已经发现了许多转录因子结合位点,正是在这些转录因子复杂的调控机制之下,SIRT1的转录活性维持在一个相对稳定的水平。其中,关于组蛋白甲基化对于SIRT1的转录调控机制方面的研究几乎没有什么报道,因此我们想看一下组蛋白甲基化对于SIRT1的转录水平是否存在一定影响,而这一机制对于心肌缺血所导致的心梗模型又有怎样的作用,这些问题都是我们需要探讨的。 方法:我们分别在人胚胎肾细胞(293)以及大鼠的心肌细胞(H9C2)中给予MTA(广谱的甲基化转移酶抑制剂)处理,通过实时定量PCR和荧光素酶报告基因活性分析实验检测MTA对于SIRT1的mRNA和转录水平的影响。通过Western blotting检测MTA对于SIRT1蛋白水平影响。利用报告基因实验我们筛选出潜在的甲基化转移酶SUV39h。为了进一步验证其特异性,我们购买了SUV39H的特异性抑制剂chaetocin。当上述细胞给与该抑制剂处理后,同样利用报告基因、RT-PCR以及Western blotting检测了SIRT1转录表达水平。在H9C2细胞中,利用siRNA将SUV39h1和SUV39h2敲除之后检测SIRT1的转录水平。最后,我们通过体内和体外实验构建大鼠心肌梗死模型来检测该药物对于心梗是否具有一定的保护作用。 结果:在心肌细胞中,无论是给予MTA还是Chaetocin处理,SIRT1的转录水平以及表达水平均有所上升,通过筛选我们发现它们作用靶点可能是组蛋白甲基化转移酶SUV39h,该家族有2个成员。利用siRNA技术,我们发现只有SUV39h2发挥作用。在体内和体外模型中,我们发现给予Chaetocin处理的确能够缓解心肌缺血所造成的心肌梗死。我们的研究将组蛋白的甲基化水平与心肌梗死症状联系在一起,进一步证明了SIRT1在保护心脏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.
【学位授予单位】:南京医科大学
【学位级别】:硕士
【学位授予年份】:2012
【分类号】:R392

【共引文献】

相关博士学位论文 前2条

1 曹春雨;热应激细胞中CIITA基因的表观遗传调控研究[D];北京协和医学院;2012年

2 夏骏;肺部免疫反应和血管重塑中重要转录事件调控机制的研究[D];南京医科大学;2013年

相关硕士学位论文 前1条

1 孔小岑;脱乙酰酶在巨噬细胞和平滑肌细胞中对CIITA转录活性的调节[D];南京医科大学;2010年



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