【作者】 谢雄伟；

【导师】 庄汉屏；

【作者基本信息】 中南大学 ， 老年医学， 2009， 博士

【摘要】 背景由于再生能力有限,心肌梗死后存活的心肌细胞不能修复梗死区,局部心肌组织缺血、缺氧,心肌细胞大量坏死,纤维组织增生,疤痕组织形成,心室重塑,导致心功能下降。BMMSCs移植可以增加梗死区肌细胞数量,改善心功能,血管内皮细胞生长因子在心肌梗死急性期的表达增加,促进梗死区新生血管形成。Notch信号是细胞与细胞之间直接作用的主要信号通路之一,并对多细胞生物中细胞命运的决定起关键作用。ICN是Notch的活性形式,通过载体介导将ICN转染细胞就可以在没有配体存在的情况下激活Notch信号,为研究Notch信号的生物功能提供了方便。目的本研究旨在观察激活Notch信号的BMMSCs移植到梗死心肌后毛细血管密度及心功能改善情况。目前转染ICN基因的BMMSCs移植对心肌梗死后心脏重构的影响还不是很清楚。本研究观察逆转录病毒介导的ICN基因转染BMMSCs后ICN基因的表达,观察逆转录病毒介导的ICN基因转染的诱导BMMSCs植入梗死心肌后对心脏重构的影响,BMMSCs能否在梗死区存活、向心肌细胞方向分化,以及梗死后心功能和心肌灌注是否得到改善,并探讨其机理,为BMMSCs转染基因治疗缺血性心脏病提供实验依据。方法1.用密度梯度离心法分离大鼠BMMSCs,体外培养、5-氮胞苷诱导,用相差显微镜、电子显微镜、免疫细胞化学方法和流式细胞仪等对诱导细胞进行鉴定。2.通过逆转录病毒介导的基因转移方法转染ICN激活BMMSCs的Notch信号通路,用RT-PCR法测定转染后ICN在BMMSCs的表达。3.结扎大鼠冠状动脉前降支建立大鼠急性心肌梗死模型,超声心动图观察心肌梗死前、后心功能变化,并观察结扎前、后心电图变化。4.选取的80只近交系Wister大鼠随机分为假手术对照组10只(A组)、建立急性心肌梗死(AMI)模型组70只,成功制成心肌梗死模型并成活60只,建立模型后随机分为MI模型组(B组)、培养液移植组(C组)、激活Noth信号的BMMSCs移植组(D组)、BMSCs移植组(E组),每组15只。移植前2小时,培养细胞用DAPI标记。结扎冠状动脉后2周,均经前次手术经路开胸,分别向梗死区注射容积为0.1ml的DMEM培养液、BMSCs、激活Noth信号的BMMSCs,假手术组则在左前降支周围心前壁多点注射0.1ml生理盐水。分别在造模后1天、28天行心脏超声心动图检查比较心功能变化。移植后28天处死大鼠,心脏标本测量比较形态变化;心脏标本切片行荧光显微镜检查定位移植细胞存活情况;免疫组化检查Ⅷ因子抗体表达计数毛细血管密度,VEGF表达情况,检查结蛋白,肌钙蛋白I等抗体表达鉴定移植细胞分化,并比较胶原蛋白、基质金属蛋白酶表达变化。结果1.成功分离出大鼠BMMSCs,流式细胞仪检查提示培养细胞表达CD29,Flk-1,CD117等抗体。2.成功地通过逆转录病毒介导的基因转移方法将ICN转染BMMSCs以激活了BMMSCs的Notch信号通路,用RT-PCR法测定转染后ICN在BMMSCs的表达。3.成功制备大鼠急性心肌梗死模型。阻断冠状动脉血流后,心电图示肢体导联ST段压低,心率加快,出现室性早搏;术后ST段弓背抬高,术后8小时ST段下降,QRS波增宽,出现病理性Q波;术后24小时彩色多普勒超声检查见左心室心腔扩大,前壁局部心肌运动障碍,左心室射血分数术后(39.67±2.33)%较术前(62.35±3.49)%及假手术组(60.52±4.75)%明显下降(p＜0.01);术后4周的大鼠心脏标本HE染色可见左心室前壁局部结缔组织增生,心肌细胞明显减少,提示心肌梗死区形成。4.细胞移植组心脏标本HE染色示左心室前壁有结缔组织增生,形状不规则,其中有成纤维细胞,有异源性细胞;心梗模型组、DMEM培养液移植组标本HE染色可见疤痕组织,未发现异常细胞。假手术组未发现疤痕组织和异常细胞。在细胞移植组心脏标本中发现标记的荧光细胞,在心梗模型组、DMEM培养液移植组心脏标本中未发现标记细胞,在假手术组心脏标本中未发现标记细胞。移植细胞组结蛋白染色阳性,肌钙蛋白I染色阳性,提示移植的细胞向心肌样细胞分化趋势,并且激活Notch信号的BMMSCs移植组抗体阳性表达明显高于单纯BMMSCs移植组。心脏彩色多普勒超声检查发现细胞移植后4周,心肌局部运动功能障碍消失,心腔缩小,左心室射血分数升高。统计学分析表明,激活Notch信号的BMMSCs移植组大鼠心功能与BMMSCs移植、心梗模型组和DMEM培养液移植组相比,明显改善。与BMMSCs移植、心梗模型组和DMEM培养液移植相比,激活Notch信号的BMMSCs移植组大鼠心脏的心肌梗死范围明显减小,心室壁厚度明显增加,毛细血管密度明显增加,VEGF蛋白表达明显增多,I型胶原抗体、基质金属蛋白酶-1抗体表达明显减轻。结论激活Notch信号的BMMSCs移植及单纯BMMSCs移植细胞可在心肌内存活,并分化为心肌样性细胞,高效表达VEGF蛋白,梗死区新生血管形成增加,心脏胶原、基质金属蛋白酶合成降低,心脏重构减轻,心功能明显改善。激活Notch信号的BMMSCs移植治疗心肌梗死是有效的。更多还原

【Abstract】 Background Viablec ardiomyocytes after myocardial infarction(MI) are unable to repair the necrotic myocardium due to their limited capability of regeneration.Coronary artery occlusion caused myocardial infarction,followed by cardiomyocytes death,fibroplasia,scar formation and cardiac remodeling due to ischemia.Heart function abruptly decreased.Transplantation of bone marrow mesenchymal stem cells (BMMSCs) increased the number of myogenic cells in myocardial infarction tissue and improved heart function.Level of serum vascular endothelial growth factor(VEGF) increased at acute phase of myocardial infarction.It may enhance angiogenesis around myocardial infarction tissue.Notch signal is one of the main direct action signal path between cell and cell,and is key point to determine cell fate of organisma multicellularis.The Intracellular Domain of Notch(ICN) is activity style of Notch signal,Notch signal is activated without ligand through transfection of ICN by carrying agent mediated.It provide convenience to study and research for biological function of Notch signal.Purpose we designed the experiment to probe the efects of capillary density and heart function through BMMSCs transfected ICN gene on cardiac remodeling after myocardial infarction.At present,The influence of BMMSCs transfected ICN gene on cardiac remodeling after myocardial infarction on the heart reconstitution is not very clear.we designed the experiment to probe the influence of the heart reconstitution through BMMSCs transfected ICN gene on cardiac remodeling after myocardial infarction. ntroversial.In order to investigate whether the implantation of BMMSCs results in sustained engraftment and myogenic differentiation,improves the postinfarction left ventricular(LV) perfusion and cardiac function.This research would provide experimental data for the treatment of ischemic heart disease with BMMSCs transferred with gene.Methods 1. BMMSCs were isolated from inbred line Wister rat’s tibia and fibula with density gradient centrifugation,cultured in vitro,and induced with 5-azacytidine.The phase-contrast microscope,electron microscope, immunohistochemistry stain,and flow cytometry were applied to identify the induced cells.2.Our lab had constructed BMMSCs with Notch signal is activated without ligand through transfection of ICN through retrovirus-mediated gene transfer.We detect the express of ICN in BMMSCs after ICN transfection through Retroviridase polymerase chain reaction(RT-PCR).3.Acute myocardial infarction model of rats was constructed by occlusion of left coronary anterior descending branch.Chang of heart function was compared between post MI and before MI by echocardiogragm and electrocardiogram.4.80 inbred line Wister rats was random divided sham operation control group(A group,10 rats) and construction acute myocardial infarctionsurvived(AMI) group(70 rats),60 rats survival from operation were randomly and equally assigned to myocardial infarctionsurvived group(B group,15 rats),culture fluid transplantation group(C group,1 5 rats),BMMSC s activaed Notch signal transplantation group(D group,15 rats) and BMMSCs transplantation group(E group,15 rats).Cultuerd cells were labeled with DAPI 2 hours before cel ls transplantation.2 weeks after ligation of left coronary anterior descending branch,0.1ml volume of induced BMMSCs transferred with ICN,induced BMMSCs without transfection of ICN,culture fluid,and physiological saline were injected into the area of myocardial infarction,respectively.Echocardiography examinations were performed to evaluate heart function at day 1,28 after operation,respectively.Rats were sacrificed 28 days after operation,heart samples were fixed with formalin for pathologic examination. Morphologic change of heart samples were assessed by gross examination.Transplanted cells were located by fluorescence microscopy examination.Capillary density,which was identified by expression ofⅧfactor antibody,was counted in hearts,Desmin and troponinⅠantibody stain were used to identified the difeerntiation of transplanted cells.Expression of collagen and MMPs in rat’s hearts was evaluated as well.Results 1.BMMSCs of rats was successfully segregated.Flow cytometry showed that cultured cells were positive for anti-CD29 antibody,anti-Flk-1 antibody,and anti-CD117 antibody.All of which confirmed that the induced BMMSCs differentiated into the myogenic cells.2.successfully constructed BMMSCs with Notch signal is activated without ligand through transfection of ICN through retrovirus-mediated gene transfer.We detect the express of ICN in BMMSCs after ICN transfection through RT-PCR.3.Myocardial infarction of rats was successfully constructed.ECG showed that ST segment was depressed and heart rates was faster when coronary artery was obstructed,ST segment was elevated and QRS complex was wider,and ST segment was depressed and Q wave appeared 8 hours later.Left ventricle was enlarged,local myocardial dyskinesis was found,and left ventricle Ejection Fraction(EF) decreased obviously by echocardiogram.Left ventricle EF of the operated rats(62.35±3.49)%, 24 hours after operation was lower than that of the rats before operation(39.67±2.33)%and sham operation rats(60.52±4.75)(p＜0.01).HE stain of heart samples collected 4 weeks after operation displayed local scar tissues devoid of cardiomyocytes,suggesting the formation of myocardial infarction.4.HE stain showed there was irregular scar tissues in anterior wall of left ventricle in cell D and E group,where allogenic cells(fluorescent cells) were found.Scar tissues but not allogenic cells were found in A group.Transplanted cells were positive for desmin and troponinⅠantibody.Echocardiogram demonstrated that local myocardial dyskinesis disappeared,volume of left ventricle got smaller,and left ventricle EF was elevated.Results of statistics analysis were as follows:heart function of rats in D group was beter than that of E group, B group,and C group.Infarcted areas and thickness of rats in D group were beter than that of E group,B group,and C group.Capillary density in D group was much more than that of others groups.Expression of type 1 collagen and MMP-1 in rats in induced D group was weaker than that of others groups.Conclunions Transplanted cells were alive in infarct myocardium and diferentiated into myogenic cells.Transplantation of BMMSCs transferred with ICN into myocardial infarction increased angiogenesis in infarct myocardium,decreased synthesis of collagen and matrix metalloproteinase,alleviated cardiac remodeling,and improved heart function.Transplantation of BMMSCs transferred with ICN was useful and safe for the treatment of myocardial infarction.更多还原