自体内皮前体细胞移植促缺血心肌血管新生的实验(英文)

Implantation of autologous murine endothelial progenitor cells promotes neovascularization in ischemic myocardium

背景:循环中内皮前体细胞在一定的条件下可向内皮细胞转化,而且可进一步形成血管。目的:探讨从外周血中获取的内皮前体细胞自体移植后促进心肌缺血区域血管新生的可行性和有效性,为冠状动脉硬化性心脏病患者的治疗提供新的细胞移植学方法。设计:完全随机对照实验。单位:青岛儿童医院心脏中心。材料:选用雄性SD大鼠60只,清洁级,体质量(340±20)g,由青岛实验动物中心提供。实验动物随机分为两组:实验组和对照组,每组30只。两组又按内皮前体细胞注入后2,4,8周3个时间点进行观察,每个时间点10只。实验过程中对动物的处置符合动物伦理学标准。方法:实验于2003-05/2004-09在青岛医药生物科技重点实验室完成。将实验组动物麻醉后,抽取动物的外周动脉血,应用密度梯度离心法获取单个核细胞。应用加入血管内皮生长因子和碱性成纤维细胞生长因子的特定培养基培养后,获得CD31、CD34、FIk-1和血管性血友病因子免疫荧光染色阳性的内皮前体细胞。结扎SD大鼠冠状动脉左前降支,建立急性心肌梗死动物模型,然后将得到的自体细胞重新植入缺血心肌局部区域。对照组注入细胞培养液,其余步骤与实验组相同。分别于结扎后2,4,8周过量麻醉处死所有动物,制作心脏组织切片。主要观察指标:①进行HE染色后光镜下观察心肌基本结构变化情况。②组织切片经Ⅷ因子免疫组化染色后,应用德国ZEISS(蔡司) Axiotron图像分析仪分析视野内Ⅷ因子阳性内皮细胞数量和微血管密度。结果:纳入的60只SD大鼠全部进入结果分析。①对照组心肌组织结构较为杂乱,心肌细胞多为被胶原组织和成纤维细胞所替代,梗死边缘区心肌细胞呈现不规则形状,部分细胞明显肥大。与对照组比较,移植组心肌胶原纤维融合较少,组织排列结构更为有序;移植区域微血管密度明显增高。②内皮前体细胞注入后第2,4和8周组心肌缺血区域微血管密度明显高于对照组的相应时间点,差异有显著性意义(P<0.01)。实验组缺血区域微血管密度随时间推移呈增长趋势,差异有显著性意义(P<0.05)。但是对照组组内比较,差异无显著性意义(P>0.05)。结论:通过一定的体外分离、定向分化、培养、扩增途径,可以从外周血获得较为纯化的内皮前体细胞;内皮前体细胞移植对局部梗死心肌组织结构有一定的保护作用,并可促进血管新生。

BACKGROUND： Under certain condition, circulating endothelial progenitor cells （EPCs） can differentiate into endothelia cells, and further participate in angiogenesis. OBJECTIVE： The goal of this study was to investigate the feasibility and efficacy of peripheral blood-derived EPCs in promoting angiogenesis in the ischemic myocardium, in order to provide a new cell implanting method for the treatment of coronary heart disease. DESIGN： A randomized controlled experiment. MATERIALS： Sixty male Sprague-Dawiey （SD） rats, of clean grade, eighing （340±20） g, were provided by Qingdao Laboratory Animal Center. These animals were randomly divided into 2 groups with 30 rats in each： experimental group and control group. In each group, ten rats were separately observed 2, 4 and 8 weeks after EPCs being injected. The protocol was conducted in accordance with animal ethics guidelines for the use and care of animals. METHODS： This study was carried out in the Qingdao Key Laboratory of Medical Biological Technology between May 2003 and September 2004. After SD rats in the experimental group were anesthetized, peripheral blood was taken. Mononuclear cells were harvested by density gradient centrifugation. CD31, CD34, FIk-1 and von Willebrand disease factor immunofluorescence staining positive EPCs were harvested by adding the defined media of vascular endothelial growth factors and basic fibroblast growth factor. Myocardial ischemia was induced by ligation of murine left anterior descending coronary artery. Autologous EPCs isolated from the peripheral blood of each animal were infused to ischemic myocardium. In the control groups,cell culture media were infused, and the other procedures were the same as those in the experimental group. Two, four and eight weeks after ligation, all animals were sacrificed by overdose anesthesia, and heart tissue sections were made. MAIN OUTCOME MEASURES： ①After haematoxylin-eosin staining, myocardial structure changes were observed under an optical microscope. ②Cells positive for factor VIII were numbered and then to calculate the total number of cells in each visual field for evaluating microvessel density with a German ZEISS Axiotron image analyzer. RESULTS： All the 60 SD rats were involved in the final analysis, without deletion. ①Myocardial structure was relatively disarrayed in the control groups, with collagens and fibroblasts substituted for cardiomyocytes. In the marginal infarct areas, cardiomyocytes were irregular and partial cells were significantly hypertrophied. As compared with control group, myocardial collagen fibers fused less, and tissue structure got more disarranged in the experimental group. Microvessel density in the implanted area was obviously increased. ②In the 2^nd, 4^th and 8^th weeks after EPC infusion, microvessel density at the ischemic myocardium was significantly higher than that at the corresponding time point in the control group （P 〈 0.01）. In the EPCs groups, microvessel density tended to statistically increase with time going （P 〈 0.05）. No prominent difference was observed in the three control groups （P〉 0.05）. CONCLUSION： Relatively purified EPCs can be obtained by certain procedure of isolation and culture from rat peripheral blood. Intramyocardial implantation of autologous EPCs promotes microangiogenesis and has a protective effect on ischemic myocardial tissue.