摘要
背景:应用骨髓间充质干细胞移植修复坏死心肌,改善心脏功能已受到广泛关注。最近有报道指出激光具有促进骨髓间充质干细胞治疗心肌缺血的作用。目的:观察兔自体骨髓间充质干细胞移植联合激光心肌血运重建治疗急性心肌缺血的效果。设计、时间及地点:随机对照动物实验,于2006-01/07在安贞医院外科实验室完成。材料:成年雄性新西兰大白兔32只,随机分为4组:模型对照组、细胞移植组、激光组、联合组,8只/组。方法:造模前3d,经兔双侧胫骨穿刺抽取骨髓,经密度梯度离心和换液法分离纯化骨髓间充质干细胞,并行BrdU标记。各组兔均在全麻下结扎冠状动脉前降支建立急性心肌缺血模型,2周后,模型对照组在标记部位分3点注射0.6mLα-MEM培养基;细胞移植组同法注射等量第4代骨髓间充质干细胞悬液,细胞总数约3×106个;激光组应用高功率CO2治疗仪进行激光心肌打孔3个,功率为5J;联合组在标记点先行激光打孔,再在孔道周围心肌中层注射等量细胞悬液。主要观察指标:治疗4周后取出心脏,采用抗BrdU及抗肌钙蛋白T双重免疫组化染色检测骨髓间充质干细胞在梗死区心肌存活及分化情况,采用抗CD146免疫组化染色测定毛细血管密度,通过超声测定左室射血分数,通过计算心尖部位位移趋势及运动速度(应变)评价心肌阶段运动功能。结果:细胞移植组、联合组均检出岛状分布的双染阳性细胞,胞核呈黑色、胞浆呈棕色。与模型对照组比较,细胞移植组、激光组、联合组治疗区血管密度均显著增加(P均<0.05),且联合组增加幅度明显高于细胞移植组、激光组(P均<0.05),细胞移植组、激光组血管密度基本相似。与治疗前比较,治疗后4周细胞移植组、联合组左室射血分数、心尖段位移和应变值均明显改善(P<0.05),且与模型对照组、激光组比较差异有显著性意义(P<0.05)。结论:兔自体骨髓间充质干细胞可在心肌微环境内存活并分化为心肌样细胞,移植后联合激光心肌血运重建在增加心肌毛细血管密度方面优于单纯细胞移植或激光心肌血运重建治疗,在提高心脏功能方面的优势尚需进一步分析。
BACKGROUND： It has been widely paid attention that bone marrow mesenchymal stem cell （BMSC） transplantation repairs necrotic myocardium and improves cardiac function. Recent reports have shown that the laser has promotion effects on BMSCs in treatment of ischemic myocardium. OBJECTIVE： To evaluate superiority of autologous BMSC transplantation combined with transmyocardial laser revascularization （TMR） for treating acute myocardial ischemia in rabbits. DESIGN, TIME AND SETTING： The randomized controlled animal study was performed at the Laboratory of Surgery, Anzhen Hospital from January to July 2006. MATERIALS： A total of 32 adult male New Zealand rabbits were equally and randomly assigned into model control, cell transplantation, laser, and combination groups. METHODS： Bone marrow was obtained from rabbits by bilateral tibia puncture 3 days before model establishment. BMSCs were isolated, purified by density gradient centrifugation and medium change method, and then labeled with BrdU. In each group, anterior descending coronary artery of rabbits was ligated under general anesthesia to create acute myocardial ischemia models. 2 weeks later, rabbit models were injected with 0.5 mL α-MEM medium through three points in the model control group. The same volume of the fourth passage of BMSC suspension was injected into rabbits in the cell transplantation group, about 3× 10^6 cells. Rabbits in the laser group were treated with TMR using high power CO2 apparatus, perforating 3 holes, at the power of 5 J. Rabbits in the combination group were treated with TMR, and then infused with an equal volume of cell suspension in the middle layer of myocardium surrounding the hole channel. MAIN OUTCOME MEASURES： Four weeks after treatment, the left ventricular myocardium of all sacrificing animals was harvested, and then the immunohistochemistrial sections were stained by anti-BrdU and anti-TnT to identify the survival or differentiation of BMSCs, as well as the CD146^＋ endothelial cells. Ultrasonicardiography （UCG） was performed to estimate cardiac function such as left ventricle ejection fraction, the displacement and strain of cardiac apex. RESULTS： Double stained positive cells were found, with black nuclei and brown cytoplasm in the cell transplantation and combination groups. Compared with the model control group, capillary vessel density significantly increased in the cell transplantation, laser and combination groups （P 〈 0.05）, and the increase was significantly higher in the combination group than in the cell transplantation and laser groups （P 〈 0.05）. The vascular density was similar between the cell transplantation and laser groups. Compared with pretreatment, ejection fraction value and the displacement or strain of cardiac apex was significantly improved at 4 weeks after treatment in the cell transplantation and combination groups （P 〈 0.05）. There was significant difference in improvement of cardiac function compared with the model control and laser groups （P 〈 0.05）. CONCLUSION： Autologous BMSCs from rabbits can differentiate into myocardium-like cells in myocardial microenvironment. Combining autologous BMSCs with TMR therapy has benefits of increasing myocardial capillary vessel density compared with BMSCs or TMR alone. The superiority in elevating cardiac function deserves further analysis.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第19期3759-3764,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
