机械打孔复合缓释支架联合干细胞移植对急性心肌梗死的作用研究

【作者】 栾云；

【导师】 刘晓程； 赵春华； 张杰民；

【作者基本信息】 中国协和医科大学 ， 外科学， 2010， 博士

【摘要】 第一部分：骨髓间充质干细胞移植治疗急性心肌梗死的作用研究实验目的：本实验应用外科手术方法对中华小型猪建立急性心肌梗死模型,通过直接注射方式将体外培养的骨髓间充质干细胞分多点注射入梗死区域心肌,饲养动物3个月后观察心脏功能改善作用,及干细胞在梗死心肌内的存活转化情况。实验方法：中华小型猪髂前上棘抽取骨髓后,体外分离培养扩增得到骨髓间充质干细胞并进行标记。结扎冠状动脉建立猪急性心肌梗死模型,随机将实验动物分为3组,每组6只。第一组心肌梗死组：单纯进行冠状动脉结扎建立急性心肌梗死模型；第二组生理盐水对照组：用生理盐水代替骨髓间充质干细胞注射入心肌梗死部位;第三组干细胞移植组：采用直接注射法将标记好的骨髓间充质干细胞分多点移植入心肌梗死区域。饲养动物3个月后检测心脏功能改善情况,并通过免疫荧光观察移植干细胞的幸存与转化情况。实验结果：实验动物手术操作3个月后,骨髓间充质干细胞移植组与模型对照组相比心脏射血分数(EF%)明显改善(分别为50.2±5.3和58.9±1.5；P<0.01),心肌灌注缺损百分数(MDP%)显著减少(分别为1.95±0.22,和-1.75±0.19；P<0.05)。免疫荧光及免疫组织化学检验结果证实移植骨髓间充质干细胞仍然存在,并且部分实验显示干细胞具有分化为心肌细胞及血管内皮细胞的迹象。结论：通过直接注射法将同种异型来源的骨髓间充质干细胞移植入急性心肌梗死部位后,骨髓间充质干细胞在体内存活可达3个月,并能部分转化为心肌细胞和血管内皮细胞,能明显改善梗死模型的心脏功能。第二部分：心肌机械钻孔复合缓释支架联合骨髓间充质干细胞移植对小型猪急性心肌梗死模型的作用研究实验目的：近年来对缺血性心脏病的治疗取得了很大进展,其中干细胞移植是很有前途的一种治疗方法。但是由于缺乏充足的血液供应,移植干细胞的低存活率成为当今困扰临床医生的一个重要问题。本实验应用新发明的较大孔径心肌机械打孔血运重建术(Transmyocardial drilling revascularization, TMDR)复合碱性成纤维生长因子(basic fibroblast growth factor, bFGF)的肝素缓释抗凝支架置入联合骨髓间充质干细胞移植治疗心肌梗死,3个月后对心脏功能进行检测,探讨该方法作用原理。实验方法：(1)骨髓间充质干细胞培养：抽取中华小型猪乳猪髂骨处骨髓,采用密度梯度离心法分离骨髓,进行体外培养后进行表型检测,移植前应用荧光染料DiI染色。(2)建立急性心肌梗死模型,机械打孔复合缓释支架置入以及干细胞移植。实验分为4组每组6只动物,分别为对照组,干细胞移植组,机械打孔复合支架置入组,干细胞移植联合机械打孔复合支架置入组。(3)手术3个月后对各组心脏功能进行评价,采用免疫组织化学方法检测梗死区域心脏梗死面积,血管密度及凋亡细胞数量；RT-PCR方法检测梗死区域血管生成因子VEGF、vWF、TGF、IL的表达情况。实验结果：手术操作3个月后,干细胞移植联合支架置入组与其它组相比(1)心脏左心室射血分数和心肌灌注缺损明显改善(P<0.05)。(2)梗死区域心肌梗死面积减少,血管密度增加,细胞凋亡数量减少(P<0.05)。(3)促血管生成因子VEGF、vWF、TGF、IL表达增强(P<0.05)。结论：机械打孔复合支架置入术能提高干细胞移植在急性心肌梗死中的治疗作用,改善猪急性心肌梗死模型心脏功能,减少心肌梗死面积,增加心肌梗死区域血管密度,为临床外科治疗心肌梗死提供了新的治疗思路。更多还原

【Abstract】 Objective:In this study, application of surgical methods on the chinese mini-swine model of acute myocardial infarction by direct injection of mesenchymal stem cells into myocardial infarct area, after feeding animals 3 months, observed the improve of heart function, the survival and transformation of stem cells in myocardial infarcted zone.Method:Miniswine were anesthetized with intramuscular administration of ketamine followed by an intravenous drip of sodium pentobarbital, and bone marrow aspirated from the anterior iliac crest was put into a syringe containing 6000u heparin, and then, was diluted with Dulbecco’s PBS. MSCs were isolated from bone marrow with density gradient centrifugation and then resuspended in culture medium. The cells were incubated in a 95% humidified incubator at 37℃and 5%CO2. Medium was replaced every 3 days and adherent cells were retained until about 90% confluence. Before implantation, the cells were labeled with the cross-linkable membrane dye CM-Dil according to the protocol of the supplier. Briefly, 1-2×107 cells were incubated for 5 min at 37℃, and then for an additional 15 min at 4℃. After being washed with PBS, the cells were resuspended in 100μL saline and then kept on ice before transplantation.The ligation of the left anterior descending coronary artery (LAD) was performed on an AMI model. Miniswine was intubated under general anesthesia and positive pressure ventilation was maintained. The heart was fully exposed via a median sternotomy. Then, the LAD was ligated after three intermittent brief preconditioning occlusions. A bonus dose of lignocaine was given intravenously (1mg·kg-1) and then maintained at 1mg·min-1·kg-1 by intravenous dripping. After the successful establishment of the model, the miniswines were randomly divided into four groups (n=6 in each), Model group:The animals only established models of AMI; Saline group:AMI immediately followed by saline injection take the place of MSCs implantation; MSCs group:AMI immediately followed by MSCs implantation. Three months after operation, detection the cardiac function in each group and observation the surviving and transformation of bone marrow-derived mesenchymal stem cell by use Immunofluorescence microscopy. Result:Three months later, the ejection fraction (EF%) was significantly improvement in MSCs group (50.2±5.3 and 58.9±1.5; P<0.01) and the mass defect percentage(%) was decreased (1.95±0.22 and -1.75±0.19; P<0.05) in MSCs group compared with saline group importantly, Immunofluorescence and histochemical results confirm that transplanted MSCs still alive and part of the experiment appears to have differentiated into cardiomyocytes and vascular endothelial cells.Conclusion:Bone marrow-derived mesenchymal stem cell transplantation by injection method into myocardial infarction mothed, the cells could survive 3 months and differentiated into cardiomyocytes and vascular endothelial cells in sight, on the other hand, the same kinds of the opposite sex bone marrow mesenchymal stem cell transplantation can improve cardiac function infarction model. Objective:In recent years, the treatment of ischemic heart disease has made considerable progress; including stem cell transplantation is a promising treatment method. However, due to a lack of adequate blood supply, the low survival rate of transplanted stem cells becomes an important tissue to today’s troubled clinician. In this study, application of the new invention of a larger diameter of transmyocardial drilling revascularization and basic fibroblast growth factor release anticoagulant heparin stent combined bone marrow mesenchymal stem cell transplantation for myocardial infarction, Three months after operation, cardiac function was detected functional and to explore the role of the principle of the method.Method:(1) Cultured bone marrow-derived mesenchymal stem cells:extraction mini-pig suckling pig iliac bone marrow, using density gradient centrifugation of bone marrow, cultured in vitro, and tested the phenotype. Before transplantation, the cells were stained by fluorescent dyes (DiI).(2) Established model of acute myocardial infarction, transmyocardial drilling revascularization and basic fibroblast growth factor release anticoagulant heparin stent combined bone marrow mesenchymal stem cell transplantation. Experiments were divided into four groups each of six animals, respectively, the control group, stem cell transplantation group, transmyocardial drilling revascularization and heparin stent implantation group, stem cell transplantation combined with transmyocardial drilling revascularization and heparin stent implantation group.(3) Three months after operation in each group to evaluate cardiac function, using immunohistochemistry to detect the heart infarcted zone infarct size, vascular density and the number of apoptotic cells; RT-PCR method to detect the vascular endothelial growth factor (VEGF, vWF, TGF and IL) expression in infracted region.Results:Three months later, stem cell transplantation combined with stent group compared with other groups:(1) Cardiac left ventricular ejection fraction and myocardial perfusion defects significantly improved (P<0.05).(2) The vessel density was augmented, infarct size and the number of cell apoptosis was reduced (P<0.05).(3) the expression levels of von Willebrand factor (vWF), transforming growth factor-β3 (TGF-β3), vascular endothelial growth factor (VEGF), and interleukin-1beta (IL-1β) were much higher (P<0.05).Conclusions:Transmyocardial drilling revascularization and basic fibroblast growth factor release anticoagulant heparin stent implantation can improve the therapeutic effect of stem cell transplantation in patients with acute myocardial infarction, improving pig model of acute myocardial infarction cardiac function and reduce myocardial infarct size, increasing the regional vascular density of myocardial infarction. This approach provides new ideas for clinical surgical treatment of myocardial infarction更多还原