【作者】 郭峻莉；

【导师】 王国平；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 第一部分缺血再灌注损伤刺激SCF表达并诱导心脏干细胞归巢实验背景缺血再灌注损伤(Ischemia／reperfusion，I／R)是引起心力衰竭的重要原因。近年来寄居在成人心脏的c-kit阳性的心脏干细胞(cardiac stem cells，CSCs)被认为是修复损伤心肌的最合适的细胞类型。但是，缺血再灌注损伤是否可以诱导CSCs归巢至损伤的心肌尚不清楚。干细胞的的激活和迁移通常是由趋化因子调控的。KIT配基，又称干细胞因子(stem cell factor，SCF)则被认为可能是诱导CSCs迁移和归巢的重要趋化因子。我们推测，在心脏缺血再灌注损伤中SCF／c-kit信号途径可能发挥着诱导心脏干细胞迁移的重要作用，从而修复心肌损伤并改善心功能。目的通过建立心肌缺血再灌注模型，观察心肌损伤后SCF的表达变化以及其对CSCs归巢的影响。方法建立Sprague-Dawley(SD)大鼠心肌缺血再灌注模型，采用RT-PCR，western blot以及免疫组化方法检测SCF的表达变化。并于诱导心肌缺血再灌注损伤之前将BrdU标记的CSCs注射在房室交界，分别于大鼠心肌缺血后不同的再灌注时间点切取损伤部位心肌组织制作冰冻切片并进行免疫荧光染色，在荧光显微镜下计数迁移入损伤区域的CSCs数目。体外实验中，提取乳SD大鼠心肌细胞，采用模拟的缺血再灌注损伤模型诱导心肌细胞缺血再灌注损伤，通过RT-PCR和ELISA方法检测心肌细胞SCF的表达。结果RT-PCR，western blot和免疫组化的结果均显示大鼠心肌缺血再灌注损伤后的第三天，SCF的表达显著高于假手术组。缺血再灌注损伤后CSCs在缺血区域的聚集随时间延长而呈上升趋势，并于再灌注三天后的聚集具有统计学意义。心功能的结果显示，缺血再灌注三周后接受CSCs注射与未接受CSCs注射的大鼠相比较，前者的心功能明显恢复。同时体外实验的结果显示，模拟的缺血再灌注明显刺激了心肌细胞SCF的表达。结论本实验表明，缺血再灌注损伤刺激了心肌细胞高度表达SCF，并诱导CSCs归巢，进而参与损伤心肌的修复及心功能的改善。第二部分缺血再灌注损伤活化NF-κB信号而上调SCF表达实验背景目前对于心肌缺血再灌注过程中调控SCF表达的信号转导机制尚无报道。DaSilva的研究首次报道了SCF基因的第一个内含子上存在着核因子kappaB(Nuclearfactor-kappaB，NF-κB)的反应元件，提供了NF-κB激活参与SCF表达的可能。NF-κB包括P65和P50两个亚基，在正常条件下NF-κB在胞浆内与其主要的抑制蛋白IκBα结合而处于非激活状态。一旦出现刺激，IκBα将迅速被上游激酶磷酸化，并通过泛素途径发生降解。这样没有了抑制蛋白约束的NF-κB二聚体便可自由进入核内，调控下游基因的转录。目前尚未有研究阐述缺血再灌注损伤对于SCF表达的调控，在本实验中我们设想缺血再灌注损伤可能通过激活NF-κB而调控SCF的表达。目的建立心肌缺血再灌注模型，检测NF-κB的激活及其上游抑制蛋白IκBα的表达，观察其对SCF表达和CSCs归巢的影响。方法建立SD大鼠心肌缺血再灌注模型，采用凝胶电泳迁移率实验(electrophoreticmobility shift assay，EMSA)的方法检测NF-κB的表达变化，并采用western blot检测IκBα和phosphor-IκBα的表达情况。应用NF-κB的抑制剂N-乙酰半胱氨酸(N-acetyl-L-cysteine，NAC，200 mg／kg)，于诱导大鼠心肌缺血前30分钟时和再灌注开始时两次腹腔给药，观察NAC对NF-κB活化、SCF表达和CSCs归巢的影响。体外实验中，分别采用转染脂质体介导的NF-κB诱捕物寡核甘酸(NF-κB decoy oligodeoxynucleotide，NF-κB decoy ODN，1mM)和加入NAC(10mM)两种方法抑制NF-κB的激活，并观察其对SCF表达的影响。结果EMSA的结果显示缺血后再灌注30分钟时NF-κB的表达开始明显上调，至2小时达到峰值。western blot的结果显示IκBα蛋白在缺血再灌注15分钟后明显发生磷酸化而致IκBα蛋白含量下降。体内应用NF-κB的抑制剂NAC，不仅显著抑制了NF-κB的激活，并且明显减弱了SCF的表达和CSCs的归巢，并且心功能也较未注射NAC的大鼠明显下降。同时体外实验的结果显示，转染NF-κB诱捕物ODN和加入NAC两种方法均明显抑制了NF-κB的激活以及SCF的表达。结论缺血再灌注损伤通过活化NF-κB信号而刺激SCF表达。综合第一及第二部分的结果，本研究表明了缺血再灌注损伤通过激活NF-κB信号而上调SCF的表达，进而诱导CSCs归巢至损伤的心肌，参与损伤修复并改善心功能。更多还原

【Abstract】 PartⅠIschemia/Reperfusion induced cardiac stem cell homing to theinjured myocardium by stimulating stem cell factor expressionBackgroundIschemia/reperfusion (I/R) is a major cause of heart failure. Recently cardiac stem cells(CSCs) were proposed as the most appropriate cell type for heart disease therapy. However,it is still unclear whether I/R can stimulate the CSCs homing to the injured myocardium.The activation and migration of stem cells, as we know, were usually regulated by somechemoattractant factors, such as stem cell factor (SCF), the ligand of the c-kit receptor. Inthis study, we speculated that SCF/c-kit signaling pathway may be involved in themigration of CSCs, subsequently repairing the injured myocardium and contributing to theimprovement of cardiac function.ObjectiveIn this study, we utilized the I/R model to investigate the expression of SCF and thehoming of CSCs during I/R.MethodsMale Sprague-Dawley rats were subjected to a 30-min ischemia followed byreperfusion of different intervals. RT-PCR, western blotting and immunohistochemistrywere performed to detect SCF expression at mRNA and protein levels, respectively. Toassess the homing of CSCs in vivo, BrdU-labeled CSCs were injected into AV-groovebefore induction of ischemia and examined by immunofluorenscent staining in the injured myocardium after I/R. In simulated I/R (SI/R) in vitro, RT-PCR and ELISA was used todetect the expression of SCF in the cultured neonatal rat cardiomyocytes.ResultsSignificant up-regulation of SCF was occurred in the I/R group after 3 days ofreperfusion examined by RT-PCR, western blotting and immunohistochemistry respectively.From day 3 to day 6 after reperfusion, the accumulation of CSCs was significantly elevatedin the injured area, which was matched with the increased SCF expression during I/R. Theresults of cardiac function showed injection of CSCs into the AV-groove resulted in therecovery of cardiac function after I/R. Furthermore, the study in vitro revealed that SI/Rstimulated the SCF expression of cardiomyocytes.ConclusionOur results demonstrated that I/R induced CSCs homing to the injured myocardium bystimulating myocardial SCF expression and improved the cardiac function. PartⅡIschemia/Reperfusion induced stem cell factor expression viaactivation of NF-κB pathwayBackgroundNowadays, it remains entirely unknown about the signal transduction pathwaysresponsible for regulating the SCF expression during I/R. Da Silva’s study firstly identifieda NF-κB responsive element located in the first intron of the SCF gene, which provided thepossibility of NF-κB involved in the regulation of SCF transcription. NF-κB is composed ofP65 and P50. Under normal conditions, NF-κB in the cytoplasm is maintained in aninactive form because of binding to the inhibitor proteins known as IκBα, which retainNF-κB dimers in the cytoplasm. Upon stimulation, IκBαis rapidly phosphorylated,leading to the ubiquitination and subsequent degradation of IκBα. The liberated NF-κBdimers then translocate to the nucleus, where they regulate the transcription oftarget genes. Accordingly, in this study, we wondered whether NF-κB activation can reallyplay a role in regulating SCF expression during I/R.ObjectiveIn this study, we utilized the I/R model to investigate the activation of NF-κB and theexpression of IκBαduring I/R, examining its role in SCF expression as well as CSCshoming.MethodsActivation of Nuclear factor-κB (NF-κB) was determined by electrophoretic mobilityshift assay (EMSA). Western blot was performed to detect the expression of IκBαandphosphor-IκBα. 30 min prior to the induction of ischemia and at the beginning ofreperfusion, rats were twice treated with the inhibitor of NF-κB, NAC (200 mg/kg) to detect its role in NF-κB activation, SCF expression and CSCs homing. In order to confirmthe causative role of NF-κB activation in the myocardial SCF expression induced by SI/R,the cardiomyocytes were transiently transfected with the NF-κB decoyoligodeoxynucleotide (ODN,1mM) or treated with NAC (10mM) before SI/R, and thenNF-κB activity as well as SCF expression were examined in the cells.ResultsThe results of EMSA showed NF-κB activation was evident at 30 min of reperfusionand reached its maximum at 2 h of reperfusion. The level of phospho-IκBαwassignificantly elevated in the injured myocardium after 15 min of reperfusion and the totalprotein levels of IκBαwere decreased examined by western blot. Pretreatment of rats withNAC not only suppressed NF-κB activation induced by I/R but also attenuated SCFexpression and CSCs homing. Furthermore, pretreatment with NAC led to the decreasedcardiac function compared with non-NAC treatment. The study in vitro revealed treatmentwith NF-κB decoy ODN or NAC markedly contributed to the blockage of the NF-κBactivation, and subsequently attenuated the SI/R-induced myocardial SCF expression.ConclusionOur data documented that I/R markedly induced the expression of SCF, in whichNF-κB activation was greatly involved.Combination the results of partⅠand partⅡ, in this study we reported that CSCs can berecruited to repair the injured myocardium by the increased endogenous myocardial SCFvia NF-κB activation during I/R.更多还原