【作者】 张彬；

【导师】 肖献忠；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 缺血心肌恢复血流灌注过程中产生的心肌损伤,称为心肌缺血-再灌注损伤（myocardial ischemia-reperfusion injury, MIRI）,常见于冠心病等心血管疾病及体外循环下行开心直视手术、心脏移植、冠脉搭桥术、冠脉溶栓术及成形术等心血管疾病治疗过程。心肌缺血-再灌注损伤的发生涉及活性氧产生、钙超载及能量代谢障碍等机制,其结果是导致心肌细胞的死亡。核仁素是真核细胞核仁中最主要的一种磷酸蛋白质,具有多种生物学功能,可调控核糖体的生物合成与成熟,调控细胞增殖、生长、调控胚胎发生、胞质分裂、染色质复制与核仁的发生等过程。作为一种穿梭蛋白和细胞表面受体,核仁素参与多种物质在细胞核与细胞浆之间的转运以及细胞核功能的细胞外调节。近年研究表明核仁素还在细胞凋亡发生中发挥重要的调控作用。研究发现,在抗肿瘤药物、紫外线照射等所致的多种肿瘤细胞发生凋亡时,伴有核仁素蛋白的表达下调、断裂和转位改变,而核仁素过表达则导致细胞凋亡减少,并进一步发现核仁素是通过对凋亡相关分子如Bcl-2等mRNA稳定性的调控而发挥作用。然而,在心肌缺血-再灌注损伤所致的心肌细胞凋亡中,核仁素是否发挥作用及怎样发挥作用,目前尚无相关研究。本研究首先观察了大鼠缺血-再灌注损伤心肌中,细胞凋亡的发生及核仁素的表达情况；然后探讨了核仁素对过氧化氢（hydrogen peroxide, H2O2）所致大鼠心肌细胞凋亡的影响；并进一步探讨了核仁素影响凋亡发生的具体机制。主要的实验结果如下：1、核仁素在大鼠缺血-再灌注损伤心肌中的表达（1）大鼠心肌缺血-再灌注损伤模型的构建：结扎大鼠心脏冠状动脉左前降支30分钟后再灌注不同时间,血清中谷丙转氨酶（aspartate aminotransferase, AST）、乳酸脱氢酶（lactate dehydrogenase, LDH）、肌酸激酶（creatine kinase, CK）及肌酸激酶-M （creatine kinase, CK-M）明显增多,心肌组织中活性氧生成增多。这提示心肌缺血-再灌注损伤模型构建成功。（2）缺血-再灌注损伤对大鼠心肌凋亡的影响：心肌缺血30分钟后再灌注不同时间,心肌组织中caspase-3活性升高,TUNEL（terminal deoxynucleotidyl transferase mediated dUTP nick end labeling）检测发现缺血-再灌注损伤心肌中细胞凋亡发生增多,琼脂糖凝胶电泳分析发现缺血-再灌注损伤导致DNA-Ladder的形成。以上研究表明,心肌缺血-再灌注损伤导致细胞凋亡发生增多。（3）缺血-再灌注损伤对大鼠心肌中核仁素表达的影响：心肌缺血30分钟后再灌注不同时间,心肌组织中的核仁素mRNA及蛋白质水平明显降低,且核仁素蛋白发生断裂,即从110kD的全长分子断裂形成一约80kD的分子。上述结果表明心肌缺血-再灌注损伤导致细胞凋亡发生增多,核仁素表达下调并发生断裂。2、核仁素对H₂O₂所致大鼠心肌细胞凋亡的影响（1）核仁素在大鼠心肌细胞氧化应激损伤中的时空表达模式：将新生大鼠原代心肌细胞暴露于0.5mmol/L H2O2,发现核仁素的mRNA及蛋白质水平明显降低、核仁素蛋白发生断裂,且在正常情况下大部分核仁素分布在心肌细胞核,H₂O₂处理则导致核仁素向胞浆移位。（2）核仁素过表达对H₂O₂所致大鼠心肌细胞凋亡的影响：应用核仁素真核表达载体转染大鼠心肌细胞,发现核仁素过表达能明显抑制H₂O₂所致的心肌细胞caspase-3活性升高及细胞凋亡发生。（3）核仁素低表达对H₂O₂所致大鼠心肌细胞凋亡的影响：应用RNA干扰技术抑制核仁素的表达,则发现核仁素低表达能进一步升高H₂O₂所致的的心肌细胞caspase-3活性及凋亡发生。以上结果表明,在H₂O₂所致大鼠心肌细胞损伤中,核仁素表达下调并发生断裂,核仁素对H₂O₂所致的大鼠心肌细胞凋亡具有抑制作用。3、核仁素抗心肌细胞凋亡的机制研究（1）核仁素不同结构域对H₂O₂所致的大鼠心肌细胞凋亡的影响：由于在核仁素的氨基端分布有其酶解位点,我们推测氧化应激损伤时形成的80kD核仁素分子是切除了氨基端的核仁素片段,因而我们选用了核仁素氨基端（AA1-309）和核仁素羧基端（AA310-713）两个突变体来探讨核仁素抗凋亡的可能机制。将核仁素不同结构域的表达载体转染大鼠心肌细胞后,发现单纯转染对caspase-3活性及细胞凋亡发生率无明显影响；H₂O₂处理后,caspase-3活性及细胞凋亡发生率明显升高,转染核仁素氨基端片段（AA1-309）后caspase-3活性低于转染空载体心肌细胞组,而转染核仁素羧基端片段（AA310-713）后,心肌细胞caspase-3活性及凋亡发生率则明显高于转染空载体心肌细胞组。这提示核仁素氨基端（AA1-309）具有部分抗凋亡作用,而核仁素羧基端（AA310-713）则具有促凋亡作用。（2）核仁素及其不同结构域对凋亡相关基因Bcl-2、Bax表达的影响：将核仁素RNA干扰片段、核仁素全长或其不同结构域表达载体转染心肌细胞,发现抗凋亡基因Bcl-2在核仁素过表达心肌细胞中表达明显上调,在RNA干扰所致核仁素低表达的心肌细胞中明显下调,但在核仁素氨基端片段（AA1-309）或羧基端片段（AA310-713）过表达细胞中,Bcl-2表达水平与对照组细胞没有明显差异。而促凋亡基因Bax则在核仁素过表达心肌细胞中表达明显下调,在核仁素低表达心肌细胞中明显上调；Bax在核仁素氨基端片段（AA1-309）过表达细胞中的表达无明显改变,而在核仁素羧基端片段（AA310-713）过表达细胞中,Bax表达增高。上述结果提示核仁素可促进Bcl-2的表达并抑制Bax的表达,而核仁素羧基端（AA310-713）则促进Bax的表达。H₂O₂处理后,心肌细胞中Bcl-2表达下调,核仁素过表达可抑制H₂O₂所致的Bcl-2表达下调,核仁素RNA干扰可促进H₂O₂所致的Bcl-2表达下调,但核仁素氨基端片段（AA1-309）或羧基端片段（AA310-713）过表达对H₂O₂所致的Bcl-2表达下调没有明显影响；与此相反,H₂O₂处理导致心肌细胞的Bax表达上调,核仁素过表达可抑制H₂O₂所致的Bax表达上调,核仁素RNA干扰或核仁素羧基端片段（AA310-713）过表达可促进H₂O₂所致的Bax表达上调,但核仁素氨基端片段（AA1-309）过表达对H₂O₂所致的Bax表达上调没有明显影响。以上结果说明在H₂O₂所致的大鼠心肌细胞损伤中,核仁素通过增加Bcl-2表达、减少Bax表达而抑制心肌细胞凋亡；核仁素羧基端（AA310-713）则通过增加Bax表达而促进心肌细胞凋亡。（3）核仁素及其不同结构域对凋亡相关基因Bcl-2、Bax mRNA稳定性的影响：收集放射菌素D处理后的不同心肌细胞组,用real-time RT-PCR检测mRNA水平,发现正常情况下核仁素过表达对大鼠心肌细胞中Bcl-2 mRNA稳定性没有明显影响,核仁素低表达则导致心肌细胞的Bcl-2 mRNA稳定性降低；经H₂O₂处理后,大鼠心肌细胞中Bcl-2mRNA稳定性降低,但核仁素过表达能抑制H₂O₂所致的Bcl-2 mRNA稳定性下降,核仁素氨基端片段（AA1-309）过表达则对H₂O₂所致的Bcl-2 mRNA稳定性的下降没有明显影响；上述结果提示核仁素通过增加Bcl-2 mRNA稳定性,使Bcl-2表达增多而抑制氧化应激所致的心肌细胞凋亡。而核仁素过表达或低表达以及核仁素氨基端片段（AA1-309）过表达,对大鼠心肌细胞的Bax mRNA稳定性没有明显影响；但核仁素羧基端片段（AA310-713）过表达则明显增加了Bax mRNA的稳定性；经H₂O₂处理后,大鼠心肌细胞中Bax mRNA稳定性增高,核仁素过表达则明显抑制了H₂O₂所致的Bax mRNA稳定性增高。以上结果表明在H₂O₂所致的大鼠心肌细胞损伤中,核仁素通过降低BaxmRNA稳定性,使Bax表达减少而发挥抗凋亡的作用；核仁素羧基端（AA310-713）则通过增加Bax mRNA的稳定性,使Bax表达增多而发挥促凋亡作用。（4）核仁素与凋亡相关基因Bcl-2、Bax mRNA的相互作用：抽提正常或暴露于H₂O₂的大鼠心肌细胞裂解液,应用核仁素抗体进行免疫沉淀后,对沉淀物中的mRNA分子进行RT-PCR分析。结果表明,在正常大鼠心肌细胞中,核仁素可与Bcl-2 mRNA结合,心肌细胞氧化应激损伤时,核仁素与Bcl-2 mRNA结合减少；平行的Westernblot结果显示在正常情况下,主要为110kD的核仁素全长分子,H₂O₂处理后,110kD核仁素明显减少,形成大量的80kD断裂片段；以上结果提示核仁素可能是通过全长蛋白质分子与Bcl-2 mRNA结合而增加其稳定性。与此相反,在正常大鼠心肌细胞中核仁素与Bax mRNA结合很少,心肌细胞氧化应激损伤时,核仁素与Bax mRNA结合增多；结合上述平行的Western blot结果,我们推论,与Bax mRNA结合的核仁素主要是80kD的片段。正常状态下,80kD的核仁素片段较少,氧化应激损伤时,核仁素80kD片段增多。该片段可能与Bax mRNA结合而增加后者的稳定性。综上所述,核仁素是一重要抗凋亡蛋白,在心肌缺血-再灌注损伤中发挥重要保护作用。在缺血-再灌注损伤大鼠心肌及氧化应激损伤的心肌细胞中,核仁素表达下调且核仁素蛋白发生断裂,这可能是心肌缺血-再灌注损伤及氧化应激损伤发生的重要机制。在正常情况下核仁素大部分位于心肌细胞胞核,氧化应激损伤时核仁素向胞浆移位。在正常大鼠心肌细胞中,核仁素可与Bcl-2 mRNA、Bax mRNA结合；细胞氧化应激损伤时,核仁素与Bcl-2 mRNA结合减少、与Bax mRNA结合增多。氧化应激损伤时,核仁素全长蛋白分子通过与Bcl-2 mRNA结合增加其稳定性、与Bax mRNA结合降低Bax mRNA稳定性,从而增加Bcl-2蛋白表达、减少Bax蛋白表达而发挥抗凋亡作用；核仁素羧基端结构域（AA310-713）则通过增加Bax mRNA稳定性使Bax表达增多而发挥促凋亡作用。心肌缺血-再灌注损伤及氧化应激损伤可能通过减少110kD的核仁素全长蛋白质分子与Bcl-2mRNA结合,并增加80kD片段与Bax mRNA结合,使得Bcl-2 mRNA稳定性降低、Bax mRNA稳定性升高,从而导致凋亡的发生。更多还原

【Abstract】 The restoration of blood perfusion in ischemic myocardium may induce myocardial damage, a phenomenon known as myocardial ischemia-reperfusion (I/R) injury (MIRI), which usually occurs in cardiovascular diseases such as coronary artery disease and the therapies for cardiovascular diseases such as open heart surgery under extracorporeal circulation, heart transplantation, bypass operation of coronary artery, thrombolytic therapy and angioplasty. The basic mechanisms of MIRI include oxidative stress, calcium overload, energy paradox, which result in cardiomyocyte death. Apoptosis, a programmed cell death, is one of the main factors that contribute to post-reperfusion cardiomyocyte death. Nucleolin is an important phosphoprotein of the eukaryotic cell and is known to be involved in many metabolic processes besides a potential role in ribosome biogenesis and mature, which include control of cell proliferation and growth, regulation of embryogenesis, cytokinesis, chromatin replication and nucleologenesis processes, etc. As a shuttle protein and a cell surface receptor, nucleolin is involved in substance transportation between the nucleus and cytoplasm and the extracellular regulation of nucleus function. Recent studies showed that nucleolin also played an important role in regulating apoptosis occurrence. Previous studies found that the occurrence of apoptosis in a variety of tumor cells treated with drugs or ultraviolet radiation was coupled with downregulation of nucleolin protein expression, nucleolin cleavage and translocation change. Conversely, nucleolin over-expression contributed to reduced apoptosis. Furthermore, some results suggested that nucleolin functioned through regulating the mRNA stability of apoptosis-related molecules such as Bcl-2. However, it remains unknown whether nucleolin plays a role in MIRI-induced apoptosis and what the detailed mechanism is.In the present study, we observed the occurrence of apoptosis and nucleolin expression in rat myocardium with ischemia-reperfusion injury. In addition, we further explored the detailed mechanism by which nucleolin affected the occurrence and development of apoptosis by investigating the specific role of nucleolin in hydrogen peroxide (H2O2)-induced apoptosis in rat cardiomyocytes. The main results are as follows:1. Expression of nucleolin in the myocardium in rats subjected to MIRI1) Establishment of MIRI rat modelAt different time of reperfusion after the ligation of left anterior descending coronary artery (LADCA) in rats for 30 minutes, serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase (CK-M) markedly increased. The generation of reactive oxygen species (ROS) in myocardial tissue was increased. These results indicated a successful MIRI model.2) Effect of MIRI on apoptosisIn different time of reperfusion after 30 minutes of myocardial ischemia, the caspase-3 activity in myocardium was increased; terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) detection revealed that the apoptosis was elevated after ischemia-reperfusion injury; agarose gel electrophoresis showed that ischemia-reperfusion injury led to the formation of DNA-Ladder. The studies above showed that MIRI contributed to an increase in apoptosis occurrence.3) Effect of MIRI on the expression of nucleolin in rat myocardiumIn myocardial tissue at different time periods of reperfusion after 30 minutes of ischemia, nucleolin mRNA and protein levels were significantly decreased and the 110 kD nucleolin protein was cleaved to an 80 kD fragment, suggesting that MIRI led to an increase in apoptosis, downregulation of nucleolin expression and cleavage of nucleolin protein.2. The effect of nucleolin on H2O2-induced apoptosis in cardiomyocytes1) The temporal-spatial expression patterns of nucleolin in myocardial cells subjected to oxidative stress:The studies in the neonate rat cardiomyocytes incubated with 0.5 mM H2O2 showed that the nucleolin mRNA and protein levels were significantly reduced and nucleolin protein was cleaved. In addition, most of nucleolin distributed in myocardial nucleus under normal circumstances and H2O2 exposure resulted in translocation of nucleolin to the cytoplasm.2) Effects of nucleolin overexpression on H2O2-induced apoptosis in cardiomyocytesTransfecting nucleolin expression vector into myocardial cells, we found that after the H2O2 treatment, nucleolin overexpression inhibited H2O2-induced caspase-3 activation and apoptosis.3) Effects of low nucleolin expression on H2O2-induced apoptosis in cardiomyocytesUsing RNA interference technology to block the expression of nucleolin, we found that after H2O2 treatment, reduced expression of nucleolin promoted H2O2-induced caspase-3 activation and apoptosis. The results suggested that nucleolin expression was suppressed coupled with the cleavage of nucleolin protein in rat cardiomyocytes subjected to H2O2-induced injury. Nucleolin inhibited H2O2-induced apoptosis in rat cardiomyocytes.3. anti-apoptotic mechanism of nucleolin in cardiomyocytes1)Effects of different domains of nucleolin on H2O2-induced apoptosisThe amino-terminal domain of nucleolin contains proteolytic sites, so we assumed that amino-terminal domain of nucleolin was excised to form an 80kD fragment during oxidative injury, and explored the potential anti-apoptotic mechanism of nucleolin by using two mutants: N-terminal fragment of nucleolin (amino acids 1-309), C-terminal fragment of nucleolin (amino acids 310-713). Neonatal rat cardiomy-ocytes were transfected with expression vectors of different nucleolin domain. The caspase-3 activity and apoptosis rate weren’t influenced by only transfection. After H2O2 treatment, the caspase-3 activity in cardiomyocytes transfected with N-terminal fragment of nucleolin (amino acids 1-309) was lower than the cells transfected with empty vector. However, the caspase-3 activity and apoptosis rate in cardiomyocytes transfected with carboxy-terminal fragment of nucleolin (amino acids 310-713) were higher than those of group transfected with empty vector. It indicated that N-terminal fragment (amino acids 1-309) of nucleolin had less anti-apoptosis ability than full-length nucleolin, and C-terminal fragment (amino acids 1-309) of nucleolin could promote the occurrence of apoptosis.2) Effects of nucleolin and its different domains on expression of apoptosis-related gene Bcl-2 and BaxWe transfected myocardial cells with nucleolin RNA interference vector or expression vectors of different nucleolin domain, and found that Bcl-2 expression markedly increased in nucleolin-overexpressed cells and significantly reduced in low nucleolin-expressed cells. Whereas, the expression level of Bcl-2 in cells with nucleolin N-terminal fragment (amino acids 1-309) overexpression or carboxy-terminal fragment (amino acids 310-713) overexpression was not significantly different from the control group. Bax expression was significantly decreased in cells with overexpressed nucleolin and increased in cells with low nucleolin expression. But the expression level of Bax in cells with nucleolin N-terminal fragment (amino acids 1-309) overexpression was not significantly different from the control group. However, Bax expression increased in cells with the carboxy-terminal fragment of nucleolin (amino acids 310-713) overexpression. These results indicated that nucleolin could up-regulate the expression of Bcl-2 and suppress the expression Bax, and the carboxy-terminal fragment of nucleolin (amino acids 310-713) could promote the expression Bax.After treatment with H2O2, Bcl-2 expression in the cells reduced compared with the untreated group. H2O2-induced down-regulation of Bcl-2 expression was inhibited by nucleolin overexpression and promoted by nucleolin RNA interference. The overexpression of nucleolin N-terminal fragment (amino acids 1-309) or carboxy-terminal fragment (amino acids 310-713) didn’t influenced H2O2-induced down-regulation of Bcl-2 expression. Conversly, Bax expression was increased by H2O2 treatment. H2O2-induced up-regulation of Bax expression was inhibited by nucleolin overexpression and promoted by nucleolin RNA interference or nucleolin carboxy-terminal fragment (amino acids 310-713) overexpression. However, the overexpression of nucleolin N-terminal fragment (amino acids 1-309) didn’t influenced H2O2-induced up-regulation of Bax expression. Taken together, the results above indicated that in rat myocardial cells damaged by H2O2, nucleolin exerted anti-apoptotic role by increasing Bcl-2 expression and inhibiting Bax expression. The carboxy-terminal fragment of nucleolin (amino acids 310-713) promoted occurrence of apoptosis by increasing Bax expression.3) Effects of nucleolin and its different domains on mRNA stability of the apoptosis-related gene Bcl-2 and BaxWe analyzed mRNA stability of Bcl-2 and Bax genes in the rat myocardial cells incubated with Actinomycin D by using real time PCR and found that Bcl-2 mRNA stability weren’t influeced by nucleolin overexpression, but reduced by nucleolin low-expression. H2O2 exposure contributed to the reduced stability of Bcl-2 mRNA, high expression of nucleolin inhibited the reduction of Bcl-2 mRNA stability by H2O2, but Nucleolin N-terminal fragment (amino acids 1-309) overexpression had no obvious effects on H2O2-reduced Bcl-2 mRNA stability. The results suggested that nucleolin played the anti-apoptotic role during oxidative stress in cardiomyocytes by increasing Bcl-2 expression via increasing Bcl-2 mRNA stability.Bax mRNA stability of rat myocardial cells wasn’t influenced by the overexpression of nucleolin or low-expression of nucleolin or by the overexpression of nucleolin N-terminal fragment (amino acids 1-309), whereas the overexpression of carboxy-terminal fragment of nucleolin (amino acids 310-713) significantly increased the stability of Bax mRNA. H2O2 exposure led to an increase in Bax mRNA stability, which was inhibited by nucleolin overexpression. The results suggested that nucleolin played the anti-apoptotic role by decreasing Bax expression via decreasing Bax mRNA stability in rat cardiomyocytes exposed to oxidative stress. The carboxy-terminal fragment of nucleolin (amino acids 310-713) promoted apoptosis by up-regulating the expression of Bax via increasing the stability of Bax mRNA.4) Interaction of nucleolin and the mRNA of apoptosis-related gene Bcl-2 or BaxRat myocardial cells lysates were extracted and immunoprecipitated with nucleolin antibody, and the precipitate further analyzed by RT-PCR. The results showed that nucleolin could bind with Bcl-2 and Bax mRNA. Oxidative stress increased the binding of nucleolin to Bax mRNA but reduced that to Bcl-2 mRNA. Parallel Western blot results showed that nucleolin was a 110kD protein in normal rat myocardial cells and was cleaved into a fragment of about 80kD after H2O2 exposure. The study above indicated that the full length nucleolin might bind with Bcl-2 mRNA to increase its stability, however the 80kD fragment of nucleolin might bind with Bax mRNA to increase its stability.In conclusion, nucleolin is a very important anti-apoptotic protein and has important protective effect on cardiomyocytes against myocardial ischemia-reperfusion injury. Nucleolin expression was downregulated and nucleolin protein was cleaved in the myocardium with ischemia-reperfusion injury and cardiomyocytes subjected to oxidative stress, which might be one of mechanisms of myocardial ischemia-reperfusion injury. Nucleolin could bind with Bcl-2 and Bax mRNA under normal condition. Oxidative stress increased the binding of nucleolin to Bax mRNA and reduced the binding of nucleolin to Bcl-2 mRNA. Full length of nucleolin protein played anti-apoptotic role by binding with Bcl-2 mRNA to increase its stability and expression and by binding with Bax mRNA to decrease its stability and expression. nucleolin C-terminal fragment played the pro-apoptotic role by increasing the stability and expression of Bax mRNA. Oxidative stress might cause apoptosis in cardiomyocytes by increasing the binding of 80kD nucleolin to Bax mRNA to increase the stability of Bax mRNA and reducing the binding of nucleolin110kD to Bcl-2 mRNA to reduce the stability of Bcl-2 mRNA.更多还原