【作者】 蒋碧梅；

【导师】 肖献忠；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 心肌缺血及缺血-再灌注损伤导致心肌坏死和凋亡,是启动心肌重构和心力衰竭的主要原因。因此,努力减轻其所致心肌损伤成为了心力衰竭防治的关键环节。心肌缺血预适应(ischemic precondi-tioning, IP)是近年来发现的重要的心肌内源性保护现象,可明显减轻心肌缺血-再灌注(ischemia/reperfusion, I/R)损伤,即减轻缺血-再灌注所致的心律失常,改善心肌舒缩功能,缩小心肌梗死面积。深入探讨缺血预适应(IP)的心肌保护机制具有重要的科学意义,并将为心肌损伤和心力衰竭的防治提供新的思路。核仁素(又称C23)是核仁中含量最多的一种RNA结合蛋白,其已知的主要功能是结合与运输rRNA,调控核糖体的生成,调控细胞生长、增殖等。核仁素含有三个主要的功能结构域：氨基端,中心区和羧基端。核仁素的许多重要功能,如调控核糖体的生成与成熟,调控细胞生长与增殖等,主要依赖于其中心区的RNA结合结构域。核仁素是细胞内一个非常重要的具有多功能的穿梭蛋白。除了上述经典的生物学功能外,近年研究发现,核仁素在丙型肝炎病毒复制、人类免疫缺陷病毒与人副流感病毒感染宿主细胞、基因转录调控等过程中也发挥了重要作用。我们研究室以前的研究也发现活性氧诱导C2C12肌原细胞、血管内皮细胞及RAW264.7细胞凋亡时伴有核仁素表达下调。进一步采用核仁素真核表达载体及反义寡核苷酸,发现核仁素在氧化应激所致细胞损伤中发挥重要保护作用,而且发现热休克反应及热休克蛋白70可抑制氧化应激所致的核仁素表达下调及细胞凋亡的发生。上述结果提示,核仁素可能是一个重要抗损伤因子。然而,核仁素在心肌缺血预适应中是否具有及具有何种作用,目前尚不清楚。本课题在本实验室以往的工作基础上,采用大鼠心肌缺血预适应动物模型、新生大鼠心肌细胞过氧化氢预适应及缺氧预适应细胞模型,探讨核仁素在心肌缺血预适应中的时空表达模式,并探讨核仁素在心肌缺血预适应中的心肌保护作用及其机制。主要方法与结果如下：1.采用在体大鼠结扎冠状动脉前降支5min,再灌注10min,再结扎5min,再灌注10min,构建心肌缺血预适应的动物模型；采用血清酶学检测、Caspase-3活性分析及DNA“梯状”条带检测心肌受损情况；采用Real-time PCR和Western-blot检测心肌中核仁素的表达。结果显示：大鼠心肌缺血预适应导致心肌中核仁素的mRNA表达逐渐升高,12h达高峰；蛋白质水平也表现为逐渐增多,以12h、24h最明显,并明显减轻了缺血30min,再灌注3h所致的心肌损伤,即血清酶学指标下降,心肌Caspase-3活性和DNA“梯状”条带明显减少。2.原代培养新生大鼠心肌细胞经过氧化氢(100μm)预处理90min或经缺氧预处理30 min后,恢复24h,构建模拟在体心肌缺血预适应的细胞模型；采用MTT、LDH活性分析及凋亡百分率检测细胞受损情况；采用Real-time PCR和Western-blot检测核仁素的表达；采用细胞成分分离及间接免疫荧光观察核仁素的定位情况。结果显示,上述过氧化氢预适应及缺氧预适应均能明显减轻心肌细胞的氧化应激损伤(500μM H2O2)和缺氧-复氧损伤(缺氧2h,复氧12-24h)；在上述过氧化氢预适应及缺氧预适应心肌细胞模型中,核仁素的mRNA表达逐渐升高,12h达高峰；蛋白质水平也表现为逐渐增多,以12h-24h最明显；而且,在正常情况下,核仁素大部分位于心肌细胞胞核,过氧化氢预适应及缺氧预适应均可导致核仁素向胞浆移位。3.采用基因转染及RNA干扰技术探讨核仁素在过氧化氢预适应所致心肌细胞保护中的作用。结果显示,核仁素过表达可增强过氧化氢预适应对心肌细胞氧化应激损伤(500μM H2O2)的保护作用,表现为LDH释放减少,细胞存活率增加,细胞凋亡减少；而核仁素低表达则明显抑制了上述过氧化氢预适应的心肌细胞保护作用。4.将新生大鼠心肌细胞暴露于过氧化氢(500μM H2O2,6h),采用核仁素抗体进行免疫共沉淀,抽提沉淀物中RNA进行大鼠基因表达谱芯片分析以及生物信息学分析,对氧化应激损伤时心肌细胞中核仁素调控的可能靶基因进行了筛选。根据大鼠基因表达谱芯片结果以及生物信息学提供的信息,初步筛选出氧化应激损伤时心肌细胞中包括Hsp32, Hsp70在内的10个可能受核仁素调控的靶基因。5.采用Real-time PCR和Western-blot技术观察到Hsp32, Hsp70在大鼠心肌缺血预适应整体及细胞模型中表达明显增加,为探讨Hsp32及Hsp70在缺血预适应心肌保护中的作用,进一步采用Hsp32特异性抑制剂及Hsp70的RNA干扰片段抑制了Hsp32的活性或抑制Hsp70的表达,发现抑制Hsp32活性或抑制Hsp70表达可明显减弱过氧化氢预适应的心肌保护作用。6.采用Real-time PCR和Western-blot技术探讨了核仁素对Hsp32和Hsp70表达的调控作用。结果显示,核仁素过表达可上调Hsp32和Hsp70的表达；进一步采用mRNA稳定性分析,IP-RT-PCR、RNA-EMSA技术和荧光素酶报告基因技术探讨了核仁素对Hsp32和Hsp70基因的调控机制。结果显示,在心肌缺血预适应中,核仁素与Hsp32, Hsp70的3’UTR相结合,进一步采用荧光素酶报告基因系统检测发现核仁素可通过与Hsp32, Hsp70的3’UTR相结合,增加Hsp32和Hsp70 mRNA的,从而发挥心肌保护作用。综上所述,大鼠心肌缺血预适应及心肌细胞过氧化氢预适应及缺氧预适应可导致心肌中核仁素的mRNA及蛋白质水平表达上调,并促进其从胞核向胞浆的移位；核仁素与靶基因Hsp32, Hsp70 mRNA的3’UTR相结合,通过增加调控Hsp32, Hsp70 mRNA的稳定性,而上调Hsp32,Hsp70的表达,从而在心肌缺血预适应的心肌保护中发挥重要作用。更多还原

【Abstract】 Myocardial ischemia and ischemia/reperfusion (I/R) injury are the major causes for myocardial remodeling and heart failure. Therefore, reducing myocardial injury plays a critical role in the protection from heart failure. Myocardial ischemic preconditioning (IP) is an important endogenous protection mechanism, which was discovered to significantly reduce myocardial IR injury. Clarifying the mechanism by which IP protects myocardium has important scientific significance and may provide new ideas for the prevention and treatment of myocardial injury and heart failure.Nucleolin (C23) is the most abundant RNA-binding protein in the cell nucleolus. Its major functions are binding and transporting rRNAs, regulating the assembly of ribosomes and regulating the growth and proliferation of cells. There are three major functional domains in nucleolin:the N-terminal, the central and the C-terminal domains. The most important functions of nucleolin, such as the regulation of synthesis and maturation of ribosomes and regulation of growth and proliferation of cells, mainly depend on the RNA binding domain in the central region of the protein.Nucleolin is a very important multifunctional shuttle protein in the cell. Other than the aforementioned biological functions, in recent years, nucleolin was also reported playing very important roles in the replication of hepatitis C virus, in the infection processes of human immunodefi-ciency virus and human parainfluenza virus, and in the regulation of gene transcriptions. In previous studies, we observed the downregulations of nucleolin expression in apoptosis induced by H2O2 in C2C12 cells, HUVEC and RAW264.7 macrophages. Further studies in this lab using transfection of eukaryotic expression vector and antisense oligonu-cleotides of nucleolin demonstrated that nucleolin played important cytoprotective effects on cellular injuries induced by oxidative stress and found that the heat shock and heat shock protein 70 could inhibit the downregulation of nucleolin induced by oxidative stress as well as apoptosis. These results suggest that nucleolin may be an important anti-damage factor. However, whether nucleolin plays some roles in myocardial protection of IP or not and what the roles are, remain unclear.Based on the previous findings, in this study, we used an intact rat model of myocardial IP, and two cell models of hydrogen peroxide preconditioning and hypoxia preconditioning with neonatal rat cardiomyocytes to investigate the spatial and temporal expression patterns of nucleolin and the roles and major mechanisms of nucleolin in the myocardial protection of IP. The major methods and results are as follows:1. The myocarial protective role and expression of nucleolin in the intact rat model of myocardial IPLigation of anterior descending coronary artery in vivo was used in this study to establish the intact model of myocardial IP in rats. Briefly, episodes of ligation 5min, reperfusion 10min, and re-ligation 5min and reperfusion 10min were performed. The myocardial injuries were measured through serological enzymatic assay, analysis of caspase-3 activity and DNA ladder detection, while the nucleolin expression was detected with real-time PCR and Western-blot. The results showed that myocardial IP significantly up-regulated the expression of nucleolin in both mRNA and protein levels with the peak expression of mRNA at 12h, and that of protein at 12h and 24h, and that myocardial IP protected significantly myocardium against the injury induced by 30 min of ischemica followed by 3h of reperfusion, as indicated by the decrease of serological enzymatic indexes, myocardial caspase-3 activity and DNA laddering.2. The expression and cytoprotective effect of nucleolin in hydrogen peroxide or hypoxia-induced cell models of IP in cardiomyocytesPrimary cultures of neonatal rat cardiomyocytes were pretreated with sub-lethal hydrogen peroxide (100μM) or hypoxia to establish the cell model of IP. Briefly, the myocardial cells were pretreated with 100μM hydrogen peroxide for 90 min or hypoxia for 30 min, then recovered for 24h. The cell injuries were evaluated by MTT assay, release of LDH and detection of apoptotic percentage, while the expression of nucleolin was detected with real-time PCR and Western-blot. Sub-cellular fractionation and immunofluorescence were used to detect the cellular localization of nucleolin. The results showed that preconditioning with sub-lethal hydrogen peroxide or hypoxia, like IP, significantly attenuated the injuries of cardiomyocytes induced by oxidative stress (500μM H2O2) or hypoxia-reoxygenation (hypoxia 2h and reoxygenation 12-24h). The mRNA expression of nucleolin was gradually upregulated in these two cell models of IP with its peak expression at 12h, while the protein levels were also gradually upregulated with the maximum at 12h to 24h. Moreover, nucleolin mainly located in the nuclei of myocardial cells under normal conditions, but it translocated from the nuclei to the cytoplasm after the preconditioning with hydrogen peroxide or hypoxia.3. The role of nucleolin in the cytoprotection mediated by hydrogen peroxide or hypoxia preconditioning in neonatal rat cardiomyocytesThe effects of nucleolin overexpression and low expression on cytoprotection mediated by hydrogen peroxide preconditioning against the injury induced by oxidative stress (500μM H2O2) were determined with gene transfection and RNA interference technologies in neonatal rat cardiomyocytes. The results showed that overexpression of nucleolin enhanced the hydrogen peroxide preconditioning-induced cytoprotective effect against the injuries induced by hydrogen peroxide (500μM H2O2) in cardiomyocytes, as indicated by the decrease of LDH release and apoptotic percentage, and the increase of cell viability, while the suppressed expression of nucleolin with RNA interference, on the contrary, significantly reduced the cytoprotective effect mediated by hydrogen peroxide preconditioning. Similar results were obstained in the cytoprotection mediated by hypoxia preconditioning.4. Studies on the mechanisms of nucleolin involved in myocardial protection meiated by myocardial IP1) In order to screen the potential target genes which were regulated by nucleolin during oxidative stress, neonatal rat cardiomyocytes were exposed to hydrogen peroxide (500μM H2O2,6h). After co-immunopre-cipitation with a specific anti-nucleolin antibody, the total RNAs in the precipitate were isolated for gene profiling analysis with rat genome wide cDNA chips and bioinformatics technology. Ten candidate genes, which contain the binding sites of nucleolin, including Hsp32 and Hsp70 were primarily selected for further studies based on the results of gene profiling and bioinformatics analysis.2) The expression of Hsp32 and Hsp70 in the intact rat model and cell models of IP were found up-regulated significantly with real-time PCR and Western-blot techniques. The specific inhibitor of Hsp32 and RNA interference oligonucleotide of Hsp70 obviously attenduated the cytoprotective effect of hydrogen peroxide preconditioning against oxidative stress.3) The regulatory effects of nucleolin on gene expression of Hsp32 and Hsp70 were analyzed with transfection of nucleolin gene or its interference RNA plasmid. Analysis of mRNA stability, IP-RT-PCR, RNA-EMSA, UV-crosslinking and luciferase reporter assay were applied to further study the mechanisms by which nucleolin regulated the expression of Hsp32 and Hsp70 translationally. The results demonstrated that nucleolin up-regulated the gene expression of Hsp32 and Hsp70 by directly binding to the 3’-UTR of Hsp32 and Hsp70 mRNA and increasing their mRNA stabilities, thus played protective effect on cardiomyocytes during myocardial IP.In conclusion, the data in this study showed that mRNA and protein expression of nucleolin were upregulated by myocardial IP in the myocar-dium of rats and by hydrogen peroxide or hypoxia preconditioning in cardiomyocytes, these changes were also associated by the translocation of nucleolin from nuclei to cytosols. Nucleolin played very important myocardial protective roles by binding to the 3’-UTRs, and then upregulating the gene expression of both Hsp32 and Hsp70 in myocardial ischemic preconditioning.更多还原