【作者】 陈克彪；

【导师】 王志农； 黄盛东；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 背景和目的:随着分子生物学、现代生物技术等学科研究的发展,生物治疗在缺血性心脏病中的应用研究已取得了长足的进步。现有研究结果证明,促血管生长因子基因治疗及自体骨髓间质干细胞移植等,对急慢性缺血性心脏病均有显著的治疗作用。其中,促血管生长因子重组腺病毒以其转染细胞效率高,目的蛋白表达量大且持续时间适中等优点在同类研究中备受青睐。动物实验及临床治疗研究结果表明促血管生长因子能有效促进缺血心肌血管新生,改善血供。但进一步的研究发现基因治疗对于心功能的改善作用并不明显,其原因可能与心脏自身归巢干细胞数量较少,不足以补充坏死心肌细胞有关。心肌梗死区细胞移植是目前研究的热点,专家在充分肯定生物治疗缺血性心脏病疗效的同时,也指出了该方法目前存在的不足。其核心是缺血微环境对移植细胞存活率的影响及细胞移植后梗死灶局部纤维化加重的倾向。由于干细胞本身以及其子代细胞的存活、生长需要合适的微环境,当骨髓干细胞植入损伤的心肌后,虽然梗死灶边缘得到了良好的改善,但梗死灶中心区域由于局部组织缺血缺氧严重,微环境恶劣,大部分的移植干细胞并不能够有效存活,因而最终影响了治疗效果。因此,如何提高干细胞移植后细胞的存活率已成为缺血性心脏病生物治疗的一个新的研究热点。此外,移植细胞向成纤维细胞分化也是细胞移植治疗所不容忽视的潜在制约因素。由于干细胞具有多向分化能力,移植至疤痕组织后,受所处微环境影响,具有明显向成纤维细胞分化的倾向。从某种程度上讲,植入细胞甚至有进一步加重纤维疤痕形成的可能。已有的研究结果表明,肝细胞生长因子（Hepatocyte growth factor,HGF）除具有促进内皮细胞修复和血管新生功能外,还可使梗死区域细胞凋亡率显著下降。更为重要的是,HGF还具有明显抑制细胞纤维化的作用。作为一种兼具促血管生长,抑制细胞凋亡和纤维化的多功能细胞因子,HGF是一种潜在的改善缺血组织微环境的理想的细胞因子。此外,细胞移植方法也是制约移植效果的重要因素,以往的移植方法大都采用开胸直视下进行细胞注射,手术创伤大;移植细胞在心肌内呈“细胞岛”状聚集,不能均匀分布,呈移植细胞纤维化加重趋势,易造成心律失常,严重影响细胞移植的治疗效果。随着介入技术的发展,通过介入的方法到达心肌缺血部位进行细胞移植,可以更好的贴近临床,有望提高移植细胞的治疗效果。因此,在本研究中,我们拟采用猪缺血性心脏病为研究模型,通过冠状静脉窦路径进行Ad.HGF-MSCs治疗,在促进血管生长的同时,抑制细胞凋亡和纤维化形成,以期达到提高移植细胞存活率,缩小纤维疤痕面积的治疗效果。本课题主要研究方法:1.骨髓间充质干细胞（MSCs）的体外分离及腺病毒转染采用Percoll密度梯度分离和贴壁相结合的方法分离MSCs,培养于含10%胎牛血清的DMEM培养基内;观察培养细胞的生长状态;通过CD71、CD34、CD44及desmin的免疫组化染色和流式细胞技术进行细胞成份鉴定;电镜观察MSCs超微结构。以不同转染倍数（MOI）的Ad.LacZ转染体外培养的MSCs,通过X-Gal染色测定转染效率,确定最佳MOI值,以最佳MOI值的Ad.HGF转染MSCs,通过免疫组化染色、Western-blot和ELISA检测转染后的MSCs目的蛋白的细胞内表达及分泌情况;免疫组化检测表面标记和功能的变化。2.Ad.HGF-MSCs生物学特性的研究Ad.HGF、Ad.VEGF分别转染MSCs,用MTT法检测转染后MSCs数量变化;流式细胞仪测定MSCs细胞周期分布;缺氧环境下用Annexin-FITC和PI双标记凋亡检测MSCs凋亡情况;ELISA检测Ⅲ型胶原纤维的合成与分泌。通过上述实验来比较两者转染后对MSCs的影响。3.缺血性心脏病模型的建立及经冠状静脉窦进行细胞移植方法的探讨标准实验猪,在基础麻醉下分离右下股动脉,由动脉导管经右下股动脉进管行冠状动脉造影确定冠状动脉前降支远端,球囊封堵90min建成缺血性心脏病动物模型。由静脉导管经右颈内静脉进管到冠状静脉窦行冠状静脉造影确定心室前静脉远端行细胞移植治疗,移植CM-DiI标记的MSCs,球囊封堵3min。通过DSA冠脉造影、心脏彩超及SPECT扫描;肌钙蛋白检测、荧光显微镜下观察移植细胞存活分布情况等方法评价动物模型及细胞移植效果。4.经猪冠状静脉窦路径行MSCs^Ad.HGF移植治疗缺血性心脏病的疗效评价按实验3的标准筛选出心梗后的实验猪20只,随机分2组,每组10只。组Ⅰ为移植转染HGF腺病毒的MSCs,组Ⅱ是移植转染VEGF腺病毒的MSCs。移植细胞量为10⁸个细胞,制成2ml细胞悬液,并在移植前以CM-DiI标记细胞。通过DSA冠脉造影、心脏彩超及超声造影、SPECT扫描,比较治疗前后的Rentrop分值、射血分数（EF）、短轴缩短率（FS）、收缩期节段性室壁增厚率（RSWT）等。动物处死后进行组织切片缺血区血管计数,TUNEL染色观察心肌细胞凋亡,荧光显微镜下观察移植细胞及通过免疫组化染色观察移植细胞的分化,Westernblot检测心肌组织目的蛋白表达。本课题主要结果:1.MSCs的形态、表面标记、分化潜能及腺病毒高转染率经形态学观察和免疫组化检测,显示所培养细胞呈成纤维细胞样形态,CD71、CD44免疫组化阳性,CD34、desmin组化阴性,符合MSCs的形态和表面标记;流式细胞仪分析,CD71、CD44阳性细胞分别占73.9%、85.4%,说明采用Percoll密度梯度和贴壁相结合的方法,可分离获得较高纯度的MSCs;电镜显示MSCs核/浆比例大,含有大量吞噬小泡。MSCs体外增殖速度快,7～8天可达80%～90%融合。体外经HGF等生长因子诱导后MSCs的CD34的免疫组化转为阳性,说明MSCs向内皮系分化,其具有多向分化能力。腺病毒对于MSCs具有很高的转染效率,MOI值为50时,转染率达95%以上。Ad.HGF转染MSCs后,细胞免疫组化和Western blot检测显示细胞内有目的蛋白的表达。ELISA检测显示细胞培养上清中有目的蛋白的表达。2.Ad.HGF转染MSCs后的生物学特性HGF对缺血缺氧引起的MSCs细胞生长抑制有明显拮抗作用,在未转染HGF的情况下,缺血缺氧可诱导明显MSCs细胞凋亡,在转染HGF组,细胞凋亡明显被抑制;在转染HGF后,MSCs的PCNA表达增强并表达内皮细胞标记物,MSCs^AdHGF组的PCNA阳性表达率明显高于MSCs^AdVEGF组;HGF能有效降低Ⅲ型胶原纤维的分泌。上述结果不仅提示HGF抑制MSCs凋亡、诱导其增殖,在一定程度上HGF还有望通过降低Ⅲ型胶原纤维的分泌而抑制缺血心肌的重构从而提高心功能。从这一点上讲,HGF可能比VEGF更适宜于缺血性心脏病的生物治疗。3.猪缺血心肌模型和细胞移植方法的评价冠状动脉前降支远端封堵后静脉血检肌钙蛋白明显升高（P＜0.01）,心电图ST-T段变化明显。1周后,DSA显示全部动物模型LAD闭塞,心脏彩超示心肌明显变薄,心肌收缩功能降低,SPECT检查负荷显像、延迟显像表现为固定性缺损,经VB染色左室侧壁可见胶原纤维增生,符合缺血性心脏病模型标准。MSCs经冠状静脉窦路径移植后在缺血心肌处弥漫分布存活,无岛样聚集。4.MSCs^Ad.HGF移植治疗改善心功能的作用治疗后4周,两组彩超提示MSCs^Ad.HGF组（组Ⅰ）的EF,FS,RSWT均优于MSCs^Ad.VEGF组（组Ⅱ）（P＜0.05）,超声造影显示前者心肌灌注改善;SPECT得出的结果与心脏彩超结果一致。心脏短轴面观察及梗塞体积比计算,组Ⅰ梗塞区的百分比（3.12±1.10%）,组Ⅱ（8.45±3.73%）,有统计学差别（P＜0.05）;荧光显微镜下,组Ⅰ可见大片CM-DiI标记的移植细胞,较组Ⅱ增多;血管计数显示:组Ⅰ的血管密度为30.3±3.5个/HPF;组Ⅱ为34.5±3.7个/HPF;两组比较无统计学差异（P＞0.05）;缺血区心肌细胞TUNEL染色组Ⅰ阳性率为3.3±0.8%,组Ⅱ为9.5±1.3%,组Ⅰ的凋亡细胞阳性率明显低于组Ⅱ（P＜0.01）。说明MSCs^Ad.HGF更有效抑制心肌细胞凋亡,减少心肌梗死面积,从而保护和改善心脏功能。结论:1、采用Percoll密度梯度和贴壁相结合的方法,可分离获得较高纯度的MSCs,这些细胞具有多向分化能力;腺病毒可以安全、有效的转染MSCs,其携带的目的基因不论在体外或体内均可获得较高的表达水平。2、HGF不仅促进MSCs的增殖、抑制其凋亡的发生,且降低细胞胶原纤维的分泌。3、冠状动脉球囊封堵、经冠状静脉窦进行细胞移植治疗是一种新的有效的动物模型建立方法和细胞移植方法,可以更好的贴近临床。4、MSCs^Ad.HGF移植促进了缺血心肌局部再血管化同时,有效地降低心肌细胞凋亡和心肌纤维化,减轻左室重构的发展,保护和改善心脏功能。更多还原

【Abstract】 Background:With the development of molecular biological and modern biotechnological techniques, the research of biological treatment for the ischemic heart disease has made significant progress.Existing research results have proved that angiogenic growth factor gene therapy and autologous bone marrow mesenchymal stem cell transplantation have significant therapeutic effects on both acute and chronic ischemic heart disease.The angiogenic growth factor recombinant adenovirus can effectly transfected cells,and have large quantity of protein expression and the advantages of moderate duration,so it is thought to be good in similar studies.Animal and clinical study results show that the angiogenic growth factor can effectively promote angiogenesis in ischemic myocardium and improve the blood supply.But further research found that the improvement of cardiac function can is not significant,the reason may be associated with that the number of the heart homing stem cells is too small to replenish necrotic myocardial cells.The study on myocardial cell transplantation in the region of myocardial infarction is popular noe.The experts fully affirmed the effect of biological treatment for ischemic heart disease,but they also pointed out that the methods still have disadvantages.The core is micro-environment of ischemic affects the survival rate of transplanted cells and increase of the tendency of local fibrosis after regional cell transplantation.The survivorship and growth of stem cells,as well as their offspring cells,need appropriate micro-environment. When the bone marrow stem cells are implanted after myocardial injury,infarction marginal have been well improved,but the most of the transplanted stem cells in the core region of infarction can not survive effectively,as a result of serious local tissue hypoxia-ischemia,micro-poor environment,and eventually affect the therapeutic effect. Therefore,how to improve the survival rate of transplanted stem cells has become a new hotspot the of treatment for ischemic heart disease.In addition,the differentiation of transplanted cells to fibroblasts in the treatment should not be overlooked as the potential constraints.Because of the ability of multi-directional differentiation,stem cells have a clear tendency differentiation to fibroblasts after transplantation to the scar tissue in which the micro-environmental impacts.In a certain sense,implanted cells can even further increase the possibility of fiber scar formation.Existing research results show that hepatocyte growth factor（HGF） can not only promote the restoration of endothelial cell and the promotion of angiogenesis functions, but can also significantly decrease the apoptosis of the cell in the infarctial region. Moreover,HGF can also significantly inhibite fibrosis.As a multi functional cytokine which both can promote angiogenic growth,inhibite of apoptosis and fibrosis,HGF is a kind of can ideal cytokines which can potential improve the micro-environment of ischemic tissue.Methods:1.MSCs isolation,culture and adenovirus transfectedMSCs were harvested from pig ilium and isolated by combination of gradient centrifugation of Percoll and preplating treatment,then were cultured in EMDM medium with 10%fetal bovine serum.Cell growth curve was obtained by the continual cell counting and the ultra-structure of cells were observed by electron microscope.The cells were identified by the immunohistology staining and flow cytometer of CD71,CD34,CD44 and desmin.MSCs were incubated with HGF and other growth factors to observe their pluripotential differentiation.Transfection efficiency of adenovirus vector to MSCs were evaluated by infection with various titrations of Ad.LacZ.MSCs were transfected by Ad.HGF.HGF expression were measured by immunohistochemical staining and western blot.The concentration of secretive protein in the medium were measured by ELISA.The cells proliferation effect of secretive proteins were evaluated by proliferation of the endothelial cells.2.Bionomics of MSCs transfected by Ad.HGFMSCs was transfected by AdHGF,AdVEGF respectly,and detected the change of MSCs through MTT method.Intensity of fluorescence and cell-distribution were measured by flow cytometer.Using Annexin V-FITC and PI to mark apoptosis of MSCs. Composition and externalization of collagen fiberⅢwere detected by ELISA in order to compare diversity of them.3.Establish of pig myocardium ischemic model and transplantation of MSCsThe distal left anterior descending artery（LAD） was occluded with the balloon of an OpenSail dilatation catheter for 90 minutes to create myocardial infarction. Electrocardiography was used during the procedure to monitor the induction of ischemia, and to prevent premature death from excessive ventricular arrhythmia,guiding catheter was introduced into the ostium of the coronary sinus through the jugular vein under fluoroscopy.Next,the guiding catheter was inserted into the GCV,and advanced into the distal AIV.The cells were rotated inside the vial to achieve re-suspension before delivery, and loaded into a 10cc angiography syringe.The balloon was re-inflated,and an image was recorded（cine and still） to show the location of occlusion.To deliver the cells（all 10ml in about 10-15 sec）,the injection syringe containing the cell suspension was attached to the infusion port of the infusion catheter,and the injection was performed. Saline（2ml） was injected to flush the infusion lumen,and the balloon was left inflated for 3 minutes before deflation and withdrawal of all catheters.Blood samples were collected at 0,90,and 150 minutes after ischemia,and were centrifuged to obtain the serum for measurement of pig cardiac Troponin I（cTnI） levels.One week after cell implantation, under general anesthesia,animals were euthanized by injection of 20ml of potassium chloride into the right atrium.The hearts were excised and stainied for analysis.4.Transplantation of autologous MSCs transfected by AdHGF gene in a porcine ischemic heart model through coronary sinusOne weeks later,20 pigs were randomized（10 pigs for each group） to treatment with the MSCs transfected ex vivo by HGF group（GroupⅠ）,or MSCs transfected ex vivo by VEGF group（GroupⅡ）,The amount of total implated cells was 10⁸ and the transfected virus titrations was 10¹⁰pfu,.All cells were labeled by CM-DiI before injection.Four weeks after initiation of therapy,the animals were evaluated by regional perfusion,Rentrop score,ejection factor（EF）,fraction shortening（FS）,regional systolic wall thickening （RSWT） using DSA,echocardiography,contrast echocardiography or SPECT.After sacrificing the animals,the percentage of infarct area,vessels counting,TUNEL staining of the implanting sites were performed.The CM-DiI labeled cells were observed under fluorescence microscope and the target proteins were detected by western blot.Results:1.MSCs isolation culture and adenovirus transfected The MSCs demonstrated typical MSC fibroblastic morphology with the ability to proliferate in culture.They are positive for CD71 and CD44,while negative for CD34 and desmin by immunohistology staining. FACS analysis demonstrated that 73.9%cells expressed CD34 and 85.4%for CD 71.The cells displayed the MSCs characters under transmission electron microscope and possessed the capabilities of rapid proliferation and pluripotential differentiation.The cells showed CD34 positive when induced by HGF and other growth factors.The transduction efficiency of Adenovirus to MSCs was proved to be over 95%at MOI 50 using Ad.LacZ transfection in vitro.The MSCs were detected to express of target protein after Ad.HGF transfection by immunohistochemical staining and western blot.2.Bionomics of MSCs transfected by Ad.HGF MSCs was transfected by AdHGF and detected the change of MSCs through MTT method and found HGF may induce increment of MSCs,and antagonism apoptosis of MSCs.Intensity of fluorescence and cell-distribution were expression by flow cytometer.Using Annexin V-FITC and PI. Composition and externalization of collagen fiberⅢwere degrade compare to AdVEGEThis show that HGF was one better biotherapy to IHD than VEGF.3.Evaluation of pig myocardium ischemic model and transplantation of MSCs Of the total 40 animals,12 animals died from excessive ventricular arrhythrnia.yocardial infarction were obviously in animal mode,Di-I labeled cells seen at the site of implantation on fluorescence microscopy.TroponinI levels changed obviously（P＜0.01）.4.The improvement of perfusion and heart function after the transplantation of Ad.HGF-MSCs.4 weeks after treatment,compared with those before treatment,the perfusion were greatly enhanced in the GroupⅠ;partial enhanced in GroupⅡ（P＜0.05）.EF, FS,RSWT showed greatly improved in GroupⅠ,and the same results were reached by SPECT.The lateral infarctions were observed in the gross left ventricular（LV） cross-sections,consistent with the echocardiography results.The infarction areas in GroupⅠ（3.12%±1.10%） were much smaller than the other groups（P＜0.05）（GroupⅡ8.45%±3.73%）.GroupⅠshowed the apparent normal gross L V geometry and maintenance of wall thickness.There was successful engraftment of MSCs in all MSCs treated animals as shown by the Di-I labeled cells seen at the site of implantation on fluorescence microscopy.A large number of labeled MSCs could be identified in GroupⅠ,while only sporadic labeled MSCs were seen in GroupⅡ.Cells appeared to preferentially engraft in regions of necrotic tissue and adhere to the collagen rich matrix.The vessels counting of the ischemic area for each group were:30.3±3.5/HPF,34.5±3.7/HPF respectively.The vascular density in GroupⅠhave increased more diffusely than the other groups（P＞0.05）. For TUNEL staining,the positive rate in each group were:3.3±0.8%,9.5±1.3% respectively（P＜0.01）.Conclusions:1.High purified MSCs can be obtained by the combination of gradient centrifugation of Percoll and preplating treatment.The obtained cells showed MSCs surface markers and possessed the capabilities of pluripotential differentiation.The MSCs can be expanded rapidly in vitro;Transduction HGF gene to MSCs by adenovirus is safe and high efficient, and the target protein can be expressed in cell and secreted to surrounding at a high level in vitro or in vivo.2.MSCs was transfected by AdHGF may induce increment of MSCs,and antagonism apoptosis of MSCs.Intensity of fluorescence and cell-distribution were expression by flow cytometer.Collagen fiberⅢwere degrade compare to AdVEGF.This show that HGF was one better biotherapy to IHD than VEGF.3.The distal left anterior descending artery（LAD） was occluded with the balloon and implantation of MSCs through vena coronaria was feasible.This method consistent with clinical.4.The implantation of AdHGF-MSCs developed the collateral vessel,improved the local reperfusion,attenuate contractile dysfunctons and pathologic remodeling of the ventricular wall after infarction and significantly augmented left ventricular function.更多还原