【作者】 吴晓燕；

【导师】 崔连群；

【作者基本信息】 山东大学 ， 内科学， 2009， 博士

【摘要】 近年来冠心病、急性心肌梗塞（AMI）发病率在我国呈逐渐升高的趋势,尽管对心梗的治疗方法取得很大进步,但心梗后心室重构,最终发展为慢性心力衰竭（CHF）仍是目前国内外研究的热点。心肌细胞损伤坏死后由胶原、纤维结缔组织替代,坏死部位变薄延展导致心衰。随着药物治疗、介入技术和外科治疗水平的提高,心肌梗塞患者心功能不全的病程得到延缓,但在成功接受血运重建的心肌梗死患者中,仍有超过30%患者发生远期左室重构,引发心力衰竭。慢性心衰患者预后不良,因此对再生坏死心肌、提高患者生存率的研究已成为刻不容缓的任务。大量的实验证实,骨髓干细胞可以取代坏死的心肌细胞并建立新的血管来改善缺血心肌供血,从而明显改善心功能,临床研究较多的有骨髓间充质干细胞（bone marrow mesenchymal stem cell,BMMSCs）移植,以及采用粒细胞集落刺激因子（granulocyte-colony stimulating factor,G-CSF）进行骨髓干细胞动员两种方法。骨髓干细胞是未分化的前体细胞,可以分化为多种细胞,包括心肌细胞,动物实验研究表明,BMMSCs自体移植通过修复坏死心肌达到改善心脏功能的目的。目前研究表明,G-CSF能动员骨髓干细胞迁移至梗死部位,参与坏死心肌修复。Orlic等对大鼠的研究表明,G-CSF动员的骨髓干细胞除能分化为血管内皮细胞外,还能分化为心肌细胞、平滑肌细胞。但Norol等对狒狒心梗后使用G-CSF治疗,只是在梗死区发现有内皮细胞,没有发现新分化的心肌细胞。另外,G-CSF能增加梗死区巨噬细胞浸润,加速坏死组织的吸收,减少肉芽组织和瘢痕的形成,从而促进梗死区的愈合过程。可以认为G-CSF骨髓干细胞动员与BMMSCs移植治疗心肌梗死,其有效的细胞成份基本相同,然而目前国内外尚未有对这两种方法治疗心肌梗死后慢性心衰的效果进行比较的报道。本研究目的在于观察BMMSCs移植治疗心肌梗死后慢性心力衰竭疗效的同时,研究其可能机制,并对BMMSCs移植和G-CSF骨髓干细胞动员这两种方法进行比较,从而探讨更有效、更适合的干细胞治疗心肌梗死后慢性心力衰竭的方法。本研究分三部分:第一部分大鼠心肌梗死后慢性心力衰竭模型的制作;第二部分大鼠骨髓间充质干细胞的分离、扩增和纯化;第三部分骨髓间充质干细胞移植与干细胞动员治疗慢性心衰模型的对比研究。第一部分大鼠心肌梗死后慢性心力衰竭模型的制作目的为研究干细胞移植在治疗心衰中的价值,本研究目的在于建立大鼠心肌梗死后心力衰竭模型,完善制作方法,并进行评估,深入心衰机制的研究。方法Wistar大鼠在左冠状动脉前降支（LAD）起始部2～3mm处结扎,造成左心室大面积心肌梗死,建立心梗后慢性心力衰竭模型。观察术后大鼠表现、心电图变化。在模型建立前和建立后8周进行连续超声检查,超声心动图检查仪使用SONOS 7500型超声诊断仪,心脏探头频率为10MHz.。用二维切面超声,选取大鼠左室长轴、短轴和心尖四腔心切面结合M型多普勒超声进行检查,测量左室收缩末内径（LVDs）、左室舒张末内径（LVDd）、左室收缩末前壁厚度（LVATs）、左室舒张末前壁厚度（LVATd）、收缩末室间隔厚度（IVSTs）、舒张末室间隔厚度（IVSTd）、短轴缩短率（FS）、左室射血分数（LVEF）等指标,连续测量3次取均值;进行血流动力学检查,检测心率（HR）、左室收缩压（LVSP）、左室舒张末压（LVEDP）、计算左室压力最大上升速率（+dp/dtmax）和左室压力最大下降速率（-dp/dtmax）,并与术前和同期的对照组比较。术后8周处死,对心脏进行病理组织学检查,判断心梗区域大小。结果（1）结扎前降支即刻心电图各个导联均显示QRS波群增宽,肢导R波振幅明显升高,T波高耸,Ⅰ、Ⅱ、Ⅲ、aVR、aVL、aVF导联ST段弓背向上抬高＞0.2mv,并呈动态演变过程,1-3天后出现病理性Q波,证明模型建立成功。（2）模型组术后成活率低,大鼠出现明确的心肌梗死改变,心梗后8周超声心动图示左室前壁变薄,动度降低,左室内径明显增大（P＜0.05）;室间隔和左室前壁明显变薄（P＜0.05）,动度减弱或运动消失;FS明显降低[（29.56±7.82）vs（42.57±5.43）,（P＜0.01）]、LVEF明显减小[（40.68±13.76%）vs（80.15±4.79%）,（P＜0.01）],表明模型组大鼠心梗后心脏发生了形态和结构的变化,即心壁变薄,左心室扩大,心脏功能明显减低,出现失代偿性心衰,心梗后心力衰竭模型建立成功。（3）8周时模型组+dp/d tmax和一dp/d tmax绝对值均低于对照组（P＜0.05）,LVEDP则高于对照组（P＜0.01）。（4）病理组织学检查可见大量炎性细胞浸润,缺血部位心肌被纤维组织替代。梗死面积为19～42.3%。结论心梗后8周形成心衰模型,通过术中术后采取必要的措施,可以提高动物生存率。此方法有效且重复性好,可较理想地模拟心梗后慢性充血性心力衰竭的发生,对研究探讨慢性心衰机制有重要意义。第二部分大鼠骨髓间充质干细胞的分离、扩增和纯化目的建立一种简便、快速、实用的大鼠骨髓间充质干细胞（BMMSCs）体外分离、纯化和扩增的方法,观察其生长状况、形态学特征和生物学特性,为慢性心力衰竭模型进行干细胞移植提供种子细胞。方法采用密度梯度离心法对大鼠骨髓间充质干细胞的提取、分离、培养和体外扩增,用离心的方法分离出大白鼠的股骨骨髓,淋巴细胞分离液分离单个核细胞,经冲洗后,培养、传代,得到第三代骨髓间充质干细胞（P3）,于培养液中加入终浓度为50ug/ml的4,6二乙酰基-2-苯基吲哚（DAPI）,孵育,然后用Hanks平衡盐液洗去未结合的DAPI。利用荧光显微镜观察计数同一个视野胞核发出蓝色荧光的细胞数目,每个标本随机计数10个高倍视野取其平均值,计算二者的比值即得到DAPI的标记率。结果采用贴壁法培养BMMSCs,在接种后72小时内,大部分细胞已经贴壁,4天后,形成生长的细胞集落,并不断增大,5～12天后BMMSCs迅速生长,形成明显克隆集落,14天大部分BMMSCs融合成单层,呈漩涡状排列,0.25%的胰蛋白酶消化贴壁细胞,再经过培养5天、传代后可得到第三代骨髓间充质干细胞（P₃）,以后传代细胞,就变得宽大扁平、增殖速度减慢,有衰老迹象。DAPI荧光标记后倒置相差荧光显微镜下可观察到DAPI染色的细胞核呈蓝色荧光。分别在倒置相差显微镜和倒置相差荧光显微镜下观察细胞的分布和数目,标记效率＞97%。结论经密度梯度离心法得到的骨髓干细胞内含有较多的BMMSCs,利用贴壁培养传代方法成功对BMMSCs进行分离、培养、扩增和纯化,细胞活力好,增殖能力强。DAPI标记BMMSCs敏感性好,标记效率高,可作为标记细胞的一种有效手段,BMMSCs体外培养成功为治疗慢性心力衰竭提供了种子细胞。第三部分骨髓间充质干细胞移植与干细胞动员治疗慢性心衰模型的对比研究目的骨髓间充质干细胞移植与粒细胞集落刺激因子（G-CSF）动员自体骨髓来源的干细胞对大鼠心梗后慢性心力衰竭的治疗作用进行比较,探讨更有效、更适用的干细胞治疗心梗后慢性心衰的方法。方法建立Wistar大鼠左冠状动脉结扎方法复制心肌梗死后心衰模型,随机分为干细胞移植组、干细胞动员组和对照组。移植组（n=10）模型建立后再次开胸,于梗死区内注射经4,6-二乙酰基-2-苯基吲哚（DAPI）标记后的骨髓间充质干细胞,将细胞移植至梗死区。动员组（n=10）模型建立后开始皮下注射粒细胞集落刺激因子（G-CSF）30μg/kg·d,连续使用5天,对照组（n=10）不采取任何治疗措施。4周后采用超声心动图检查心脏功能变化,用多道生理记录仪做血液动力学测定,描记左室收缩压（LVSP）、左室舒张末压（LVEDP）、左室等容收缩/舒张期压力上升或下降最大速率（±LVdp/dtmax）以反映左室收缩与舒张功能。取心脏作病理及免疫组织化学分析,观察移植细胞分化情况和促血管生成作用。利用RT-PCR法分析各组细胞凋亡相关基因Bcl-2、Bax mRNA的表达水平。结果（1）细胞移植4周后,在移植组坏死区内未找到增殖的DAPI标记的BMMSCs,移植组和动员组坏死区内有大量的血管新生,但未发现有新生的平滑肌细胞及心肌细胞。（2） 4周后,超声检查显示对照组LVEF显著下降,移植组及动员组心脏功能显著改善,LVEF明显升高,与对照组相比均有显著性差异（P＜0.05）,但移植组及动员组间无显著性差异（P＞0.05）。（3） 4周时动员组及移植组与对照组相比+LVd p/d tmax和-LVd p/d tmax明显升高,LVEDP明显下降,均有显著性差异（P＜0.05）。（4）动员组及移植组梗塞区血管密度较对照组[（6.58±1.47）,（7.06±1.63）,vs（2.14±0.62）]血管新生明显,微血管密度均明显高于对照组（P＜0.01）,（5）原位末端标记结果示,细胞移植组和动员组心肌细胞凋亡指数显著低于模型对照组（P＜0.05）,但两者间无显著性差异（P＞0.05）。（6）与对照组相比,细胞移植组和动员组Bcl-2 mRNA表达水平明显增高（P＜0.05）,Bax mRNA表达水平降低（P＜0.05）。结论（1）骨髓干细胞移植及动员治疗大鼠心肌梗死后心衰,均能促进大鼠心肌梗死缺血区及其周边区域的毛细血管新生、减少心肌凋亡而明显改善心脏功能。（2）骨髓干细胞动员可能为慢性心力衰竭的治疗提供一种新的无创性手段。更多还原

【Abstract】 Despite the advances made in the management of acute myocardial infarction （AMI）,chronic heart failure（CHF）secondary to left ventricular remodeling following infarction continues to be a major medical problem world-wide After myocardial infarction,the infarct area is replaced by fibrocytes and collagen fibers.The nonelastic scar eventually thins and dilates,thereby causes CHF.Several therapeutic options have been developed quickly to delay the progression of ventricular dysfunction in patients with AMI.However,there are more than 30%patients with successful revascularization causes left ventricular remodeling and CHF.Patients with CHF have a poor prognosis.Emerging research in cardiac therapy is destined to regenerate infracted myocardium and enhance the rate of survival in cardiac patients.Many experimental studies have shown that bone marrow stem cells are capable of regenerating infracted myocardium and inducing myogenesis and angiogenesis; this leads in turn to amelioration of cardiac function in rats.Bone marrow mesenchymal stem cells（BMMSCs）transplantation and bone marrow stem cells mobilization with granulocyte- colony stimulating factor（G- CSF） are two strategies for the repair of infraeted myocardium which have been used in clinical study.Stem cells are undifferentiated immature precursor cells that can proliferate, self-renew,and differentiate into one or more types of specialized cells,including cardiomyocytes.Animal studies have revealed that autologous BMMSCs transplantation can repair the infracted myocardium and improve cardiac function.The present study demonstrated that G- CSF could mobilize bone marrow stem cells home to the infracted myocardium.Orlic reported that bone marrow stem cells mobilized to the mice damaged myocardium differented to myocytes, endothelial cells and smooth muscle cells.But Norol found that,in a baboons model, only endothelial cells were observed in the infracted area,without detectable differented myocytes and smooth muscle cells.G- CSF had been reported to accelerate the healing process by promoting the absorption of necrotic tissues via increase of macrophages and reduces granulation and scar tissues for the beneficial effect of CHF treatment.For the therapy of CHF,the effective cell component of bone marrow mobilization with G- CSF is the same as BMMSCs transplantation.However,the comparison between these two methods remains largely uninvestigated.We aimed to analyze the action mechanism and effect of BMMSCs transplantation on cardiac function after myocardial infarction.we compare them to explore more practical and effectively stem cells therapeutic strategy for CHF.The study include three part:（1）The experimentalstudy of the chronic heart failure model after myocardial infarction in rats.（2）Isolation,culture and proliferation of bone marrow mesenchymal stem cells in vitro.（3）Comparison of bone marrow mesenchymal stem cells transplantation with bone marrow stem cells mobilization for the therapy of chronic heart failure in rats.PART ONE THE EXPERIMENTAL STUDY OF THE CHRONIC HEART FAILURE MODEL AFTER MYOCARDIAL INFACTION IN RATSAbjective It is one of critical techniques for medical experiment to make myocardial infarction-heart failure model.The Purpose of this study is to establish and assess the model of chronic heart failure（CHF） after myocardial infaction（MI） in male Wistar rats.Methods Left anterior descending coronary artery（LAD） was ligated to induce myocardial infarction in male Wistar rats.Electrocardiograms were recorded.The control group（n=10） was as same as MI group excep for ligating LAD.Echocardiography was recorded in all experimental subjects before and after operation,using a 10-MHz phased-array transducer and SONOS 7500 echocardiography system.M-mode tracing and 2D echocardiography images were recorded from the parasternal long- and short-axis views.Left ventricular end-systolic dimensions（LVDs） and end-diastolic dimensions（LVDd）,systolic and diastolic anterior wall thickness（LVATs、LVATd）,as well as systolic and diastolic interventricular septal thickness（IVSTs、IVSTd）were measured from the M-mode tracings.For each M-mode measurement,at least three consecutive cardiac cycles were sampled.LV ejection fraction（LVEF） and fractional shortening（FS） were derived from LV cross-sectional area in 2D short-axis view.Standard formulae were used for echocardiographic calculations.All data were analyzed offline with software installed in the ultrasound system.All measured and calculated indexes were presented as the average of three consecutive measurements.Hemodynamic parameters including heart rate,left ventricular systolic pressure （LVSP）,left ventricular end diastolic pressure（LVEDP）,maximal velocity increase of pressure（+dp/dtmax） and maximal velocity decrease of pressure（-dp/dtmax） were observed.Rats in both groups were sacrificed and the histopathologic examinations were performed 8 weeks later.Results（1）The ECG ST-segments onⅠ、Ⅱ、Ⅲ、aVR、aVL、aVF leads elevated to 0.2mv persistently after ligation of left anterior descending coronary artery.After 1-3 days, the presence of Q-waves on ECG tracings indicated that myocardial infarction was induced successfully in rats.（2） Echocardiography was found that the wall thickness and motion of the LV anterior wall in model group were thinned and akinetic 8 weeks after MI.In the model group,LVDs、LVDd increased significantly（P＜0.05）;LVATs、LVATd、IVSTs、IVSTd group decreased significantly（P＜0.05）;FS[（29.56±7.82）vs（42.57±5.43）,（P＜0.01）]、LVEF[（40.68±13.76%）vs（80.15±4.79%）,（P＜0.01）]decreased significantly.（3） At 8th week,absolute value of + dp/dtmax and - dp/dtmax in model group were lower than those in control group（P＜0.05）.LVEDP were higher than those in control group.（P＜0.01）.（4） Histopathologic examinations showed there were massive inflammatory cell infiltration and fiber tissue proliferation in infracted area myocardium.The total infracted area are ranged from 19%to 42.3%.Conclusions The heart failure model forms at 8 week afterM I.The survival rate can improve with proper measures taken during and after the operation.This method can ideally induce congestive heart failure model.It is of important significance for the research of heart failure.PART TWO ISOLATION CULTURE AND PROLIFERATION OF BONE MARROW MESENCHYMAL STEM CELLS IN VITROAbjective The purpose of the study are to establish a simple,rapid,practical method and to prepare the optimal condition for the in vitro isolation,culture,purification and proliferation of rat bone marrow mesenchymal stem cell（BMMSCs）.To observe the growth and biology characterization of BMMSCs,and to provide seed cells for the therapy of CHF.Methods.Rat BMMSCs were isolated and purified by differential adhesion method. The femora bone marrow was distilled by acentric means and the monomuclear cell was isolated by lmphocyte parting liquid,then the third generation of bone marrow cells were obtained through the process of swashing、culturing and generating.The BMMSCs were incubated with 4,6-diamidino-2-phenylindole（DAPI） which density is 50ug/ml and swashed by Hanks liquid,so it labeled by the fluorescence Dye.The karyons which emit blue fluorescence in the same visual field was observed by fluorescence microscope and The labelled ratio was obtained through counting the karyons’ average.Results Primary cultured BMMSCs adhere to plastic surface within 72hours and formed the colonies of BMMSCs at 4th day.From 5th to12th day,BMMSCs grew faster and got together to form bigger colonies then before.After 14 days,the BMMSCs fused in monolayer,then subcultured by 0.25%typsine.The passage BMMSCs proliferated fast,and could be subculture about every 5 days.The third generation of bone marrow cell（P₃） was obtained.In later generation,BMMSCs grew low and multipled karyotype appeared.Rat BMMSCs were effectively purified by differential adhesion method,and grew well with good proliferation and normal morphology in medium.BMMSCs was labelled with DAPI and the labelled ratio was＞97%.Conclusions（1） Bone marrow stem cells which contained many BMMSCs can be obtained via density gradient centrifugation.（2） The BMMSCs could be isolated,cultured,expanded and purified esaily in vitro.DAPI labelling is sensitive and highly efficient,so it can be used as a method to labelling cells.（3） BMMSCs can be used as a method to gain seed cells for the therapy of chronic heart failure. PART THREE COMPARISON OF BONE MARROW MESENCHYMAL STEM CELLS TRANSPLANTATION WITH BONE MARROW STEM CELLS MOBILIZATION FOR THE THERAPY OF CHRONIC HEART FAILURE IN RATSObjective To compare bone marrow mesenchymal stem cells（BMMSCs） transplantation with bone marrow stem cells mobilized by granulocyte-colony stimulating factor（G-CSF） for the therapy of chronic heart failure（CHF） in rats,and to explore more effective and practical stem cell therapeutic strategy for CHF.Methods Myocardial infarction of left ventricle was induced in rats by ligation of left coronary artery.The rats were randomly divided into transplanted group, mobilized group and control group.In transplantation group（n=10）,BMMSCs transplantation was performed after CHF.BMMSCs labelled with DAPI were transplanted into the infarcted myocardium through intramyocardial injection 4 week later.In mobilization group（n=10）,G-CSF（30μg/kg·d） was injected subcutaneously after CHF and every 24 hours for 5 days.Control animals（n=10） did not receive any treatment after CHF.Echocardiography were performed for the evaluation of cardiac function 4 weeks later.Hemodynamic parameters including left ventricular systolic pressure（LVSP）, left ventricular end diastolic pressure（LVEDP）,maximal velocity increase of pressure per second（+ dp/dtmax） and maximal velocity decrease of pressure（-dp /dtmax） were observed.Histological examination and immunohistochemical analyses were performed to detect BMMSCs’s proliferation,differentiation and their angiogenic function 4 weeks later.The infarct size and vascular density in infarct zones were measured accordingly. The expression levels of Bcl-2,Bax and the apoptosis- associated genes were analyzed semi-quantitatively by RT-PCR.Results（1） Four weeks after cell transplantation,theBMMSCs labelled DAPI were not found in necrosis zone in transplanted group,but there were a great deal of neovascularization in necrosis zone.No regeneration of smooth muscle cells and cardiomyocytes were found in the infarcted area.（2） Echocardiography indicated that LVEF decreased significantly in control group 4 weeks later.In transplanted and mobilized group,the heart function had a great improvement and LVEF increased significantly than that in control group（P＜0.05）,and there was no significant difference between the transplanted and mobilized group（P＞0.05）.（3） Mobilized group and transplanted group had higher LV +dp/ d t max and-d p/d t max,lower LV end - diastolic pressure compared with group control group 4 weeks later（P＜0.05）.（4） Histological studies revealed that the vascular density of mobilized group and transplanted group in the infarcted area were significantly greater in comparison with control group[（6.58±1.47） vs（7.06±1.63）vs（2.14±0.62）, （P＜0.01）].（5） Both G-CSF and BMMSCs transplantation can reduce the numbers of the cardiomyocyte apoptosis（P＜0.05）,and there was no significant difference between the transplanted and mobilized group（P＞0.05）.（6） In the transplanted and mobilized group,Bcl- mRNA expression increased markedly（P＜0.05）,but Bax mRNA expression decreased evidently（P＜0.05） compared to those in the control group.Conclusions（1）This experimental study indicated that both G-CSF and BMMSCs transplantation can promote angiogenesis,reduce the numbers of the cardiomyocyte apoptosis in the infracted area and can significantly improve the heart function for the therapy of CHF.（2）Bone marrow stem cell mobilization may offer a new and non-invasive therapeutic strategy for CHF.更多还原