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1、安徽医科大学 硕士学位论文 骨髓间充质干细胞和内皮祖细胞联合移植对大鼠急性心肌梗死 的作用研究 姓名：杨宇辉 申请学位级别：硕士 专业：内科学（心血管病） 指导教师：吴继雄 20100501 安徽医科大学硕士学位论文 1 骨髓间充质干细胞和内皮祖细胞联合移植对 大鼠急性心肌梗死的作用研究 摘 要 目的： 观察经心外膜心肌内注射骨髓间充质干细胞 （mesenchymal stem cells,MSCs） 和内皮祖细胞（endothelial progenitor cells，EPCs) 移植对急性心肌梗死大鼠心功 能、心肌细胞转录因子基因、心肌细胞标志性蛋白含量、心脏梗死区毛细血管密 度的影响，
2、比较联合移植MSCs和EPCs是否比单用其中一种细胞移植效果更好， MSCs和EPCs两种细胞移植效果之间是否有差异。 方法：1.取 2 周的清洁级健康 SD 大鼠，提取骨髓，通过密度剃度离心法提取单个 核细胞，差速贴壁法和不同培养基诱导法获得 MSC 和 EPC。流式细胞术筛选，使 MSCs 和 EPCs 得以纯化。用 DAPI（终浓度为 50mg/L）标记 MSCs 和 EPCs。观 察两种细胞形态。2. 通过结扎大鼠冠脉左前降支建立心肌梗死模型。3. 实验动物 分组及细胞移植: 将已经制好心肌梗死模型的 SD 大鼠随机进行分组进行细胞移 植。A 组：MSCs 移植组 心梗模型建成后 1
3、周经心外膜心肌内注射移植已标记好 的 MSCs 细胞悬液 200ul（5x106） ；B 组：EPCs 移植组 心梗模型建成后 1 周经心 外膜心肌内注射移植已标记好的 EPCs 200ul（5x106） ；C 组：联合移植组 心梗模 型建成后 1 周经心外膜心肌内注射移植已标记好的 MSCs 细胞 5x106和 EPCs （5x106 ） ；D 组：对照组 心梗模型建成后 1w 经心外膜心肌内注射培养液 DMEM200ul。4. 细胞移植后 4 周测心脏血流动力学，随后处死大鼠取心脏标本， 作石蜡切片，荧光显微镜下观察 DAPI 标记的 MSCs 和 EPCs 并计数，用因子免 疫组化检测毛
4、细血管密度，用免疫组化测心肌肌钙蛋白 I 和缝隙连接蛋白 43，提 取心肌组织 RNA,测心肌转录因子基因。 5. 统计学处理： 所有实验数据以xs 表示， 采用 SPSS13.0 软件处理数据。多个均数间比较用单因素方差分析。若符合方差齐 性，进一步两两比较采用 Bonferroni 检验，方差不齐时两两比较采用 Tamhanes T2 法。P0.05 为差异有显著性意义。 安徽医科大学硕士学位论文 2 结果： 1.通过差速贴壁分离培养法得到较纯的间充质干细胞及内皮祖细胞。 2.MSCs 移植组、EPCs 移植组及联合移植组内均可见到 DAPI 标记的细胞，同时与对照组 相比其毛血管密度增加
5、，左心室舒张末压（LVEDP)下降，左心室收缩压（LVSP） 、 左心室压力最大上升和下降速率(dp/dtmax)、心肌特异性转录因子 GATA-4 和 VEGF 基因半定量及心肌肌钙蛋白 I 和缝隙连接蛋白 43 含量明显增高（P0.05） 。 3.细胞移植组之间相比，联合移植组上述指标改变更明显；内皮祖细胞组和间充质 干细胞移植组相比，其 VEGF 含量较高，毛细血管密度增加较明显。 结论：可应用 DAPI 标记移植的干细胞。细胞移植组心功能改善，毛细血管增多， 心肌细胞标志物增多。细胞联合移植比单用一种细胞移植效果更好。 关键词：骨髓间充质干细胞；内皮祖细胞；联合移植；急性心肌梗死 安徽
6、医科大学硕士学位论文 3 Effect of transplantation of bone marrow mesenchymal stem cells and endothelial progenitor cells on rats with acute myocardial infarction Abstract Objective: Attentive observation of intramyocardial injection of bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs) and endot
7、helial progenitor cells (endothelial progenitor cells, EPCs) transplantation on cardiac function in rats with acute myocardial infarction, myocardial cell transcription factor gene, myocardial cell markers protein content, heart infarct capillary density effects.Comparison of combined transplantatio
8、n MSCs and EPCs whether higher than that alone one of the cell transplantation, MSCs, and EPCs two kinds of cell transplantation whether there are differences between the results. Methods: 1. Take 2 weeks of clean grade healthy SD rats , extracted bone marrow , by density centrifugation extracted mo
9、nonuclear cells, differential adhesion method and the different medium induction to obtain MSC and EPC. Flow cytometry screening, so that MSCs and EPCs can be purified. With DAPI (final concentration of 50mg / L) marked MSCs and EPCs. Observed two kinds of cell morphology. 2. Through the left anteri
10、or descending coronary artery ligation in rat myocardial infarction model building. 3. Groups of laboratory animals and cell transplantation: The model of myocardial infarction have been made,SD rats were randomly grouped for cell transplantation. Group A: MSCs transplantation group 1 week after the
11、 completion of myocardial infarction model , intramyocardial injection of MSCs suspension 200ul (5x106) have been marked through the outer membrane of myocardial cells; Group B: EPCs transplantation group 1 week after the completion of myocardial infarction model , intramyocardial injection of MSCs
12、suspension 200ul (5x106) have been marked through 安徽医科大学硕士学位论文 4 the outer membrane of myocardial cells; group C: co-transplantation group 1 week after the completion of myocardial infarction model , intramyocardial injection of MSCs and EPCs suspension 200ul (5x106) have been marked through the out
13、er membrane of myocardial cells; group D : control group 1 week after the completion of myocardial infarction model , intramyocardial injection of Medium DMEM suspension 200ul through the outer membrane of myocardial cells.4. 4 weeks after cell transplantation measured cardiac hemodynamics, Then rat
14、s were killed to take the heart of specimens for paraffin sections, fluorescence microscopy observation of DAPI labeled MSCs and EPCs, and counting, With factor immunohistochemistry capillary density, measured by immunohistochemistry of cardiac troponin I and connexin 43, extracted myocardial tissue
15、 RNA, measuring cardiac transcription factor genes. 5. Statistical analysis: All experimental data with x s said that the use of SPSS10.0 software processing the data. Multiple mean comparison with the single-factor analysis of variance. If they meet the homogeneity of variance, and further using Bo
16、nferroni pairwise comparison test, If the variance is incomplete, and using Tamhanes T2 pairwise comparison method. P f0.05 for significant difference. Results: 1. Through differential adherent culture method to be more pure isolated mesenchymal stem cells and endothelial progenitor cells. 2. group A, group B and group C can see the DAPI labeled cells, compared with the control group at the same time, gross vascular density increases, left ventricular end-diastolic pres
