骨髓间充质干细胞与丹酚酸B预处理内皮祖细胞共移植对急性心肌梗死大鼠心功能及分化基因表达的影响

Effects of Salvianolic acid B-treated endothelial progenitor cell combined with bone marrow mesenchymal stem cell transplantation on heart function and gene expression in acute myocardial infraction rats

背景:由于心肌细胞自身增殖能力有限,急性心肌梗死后细胞移植是目前流行的再生心肌方法。骨髓间充质干细胞的存活受心肌微环境的影响,而内皮祖细胞参与血管新生,可为骨髓间充质干细胞提供微环境。目的:探讨骨髓间充质干细胞联合中药丹酚酸B预处理的内皮祖细胞共移植后,对急性心肌梗死大鼠心功能改善及骨髓间充质干细胞分化早期基因表达的影响。设计、时间及地点:随机对照动物实验,于2007-01/2008-03在天津中医药大学完成。材料:清洁级雄性Wistar大鼠45只,由中国医学科学院放射医学研究所实验动物中心提供。丹酚酸B为天津天士力现代中药资源有限公司产品,批号20060601。方法:分别取2只和1只大鼠,采用密度梯度离心法+贴壁筛选法分离培养纯化内皮祖细胞、骨髓间充质干细胞。剩余42只大鼠随机分为4组:假手术组9只、模型组11只、骨髓间充质干细胞组12只、共移植组10只。假手术组只开胸穿线,不结扎左冠状动脉;其余3组结扎左冠状动脉建立心肌梗死模型。结扎冠状动脉15min后再次开胸,共移植组吸取骨髓间充质干细胞悬液250μL(浓度1×1010L-1)+8mg/L丹酚酸B预处理的内皮祖细胞悬液250μL(浓度1×108L-1),于结扎区附近心肌组织分5点注射;骨髓间充质干细胞组同法注射单纯骨髓间充质干细胞悬液500μL,模型组注射等量细胞培养液。主要观察指标:采用超声心动仪测定左室功能,Real-timePCR检测心肌分化基因Nkx2.5,GATA-4的表达。结果:细胞移植4周后与模型组比较,骨髓间充质干细胞组左室结构有改善趋势,但差异无显著性意义(P>0.05);共移植组左室舒张末期内径、左室收缩末期内径、舒张末期室间隔厚度、收缩末期室间隔厚度均明显改善(P<0.05),心室收缩功能指标射血分数、心输出量均明显改善(P<0.05)。细胞移植4周后与模型组比较,骨髓间充质干细胞组、共移植组Nkx2.5及GATA-4mRNA表达均明显增加,且共移植组Nkx2.5mRNA增加幅度明显大于骨髓间充质干细胞组(P<0.05)。结论:骨髓间充质干细胞联合丹酚酸B预处理的内皮祖细胞共移植方式,可上调骨髓间充质干细胞向心肌分化过程中Nkx2.5,GATA-4基因的表达,改善急性心肌梗死大鼠心室结构,增强心功能。

BACKGROUND： Myocardial cells have limited reproductive activity. Following acute myocardial infarction, cell transplantation is a popular method of regeneration myocardium. Survival of bone marrow mesenchymal stem cells （BMSCs） is affected by myocardial microenvironment. Endothelial progenitor cells （EPCs） participate in angiogenesis, and can provide microenvironment for BMSCs. OBJECTIVE： To investigate the effect of Salvianolic acid B preconditioned EPCs combined with BMSCs on cardiac function and gene expression in rat models of acute myocardial infarction. DESIGN, TIME AND SETTING： The randomized controlled animal study was performed at the Tianjin University of Traditional Chinese Medicine from January 2007 to March 2008. MATERIALS： A total of 45 clean male Wistar rats were provided by the Experimental Animal Center, Institute of Radiological Medicine, Chinese Academy of Medical Sciences. Salvianolic acid B （Tianshili, Tianjin, China）, lot number 20060601 was used in this study. METHODS： Two and one rats were separately obtained. EPCs and BMSCs in vitro were isolated and purified by density-gradient centrifugation and adherence method. Remaining 42 rats were randomly assigned into 4 groups： sham operation group （n=9）, model group （n=11 ）, BMSCs group （n=12）, and cotransplantation group （n=10）. Rats in the sham operation group received chest open and braid, without deligation of the left coronary artery. In other three groups, the left coronary artery was ligated to establish myocardial infarction models. Following 15 minutes of coronary artery deligation, the chest was opened. Rats in the cotransplantation group were treated with 250μ L （1 ×10^10/L） BMSCs suspension ＋ 8 mg/L Salvianolic acid B-treated EPC suspension 250μL （1× 10^8/L） in five points of the myocardium surrounding the deligation region. Rats in the BMSCs group received 500μL BMSCs suspension. Rats in the model group were injected with an equal volume of cell medium. MAIN OUTCOME MEASURES： Left ventricular function was measured by ultrasonocardiograph. Expression of Nkx2.5 and GATA-4 was detected by Real-time PCR. RESULTS： Compared with the model group, left ventricular structure had the tendency to improve in the BMSCs group 4 weeks following cell transplantation （P 〉 0.05）. Left ventricular end diastolic dimension, left ventricular end-systolic dimension, interventricular septum thickness at end diastole and interventricular septum thickness at end systole were significantly improved （P 〈 0.05）, and ejection fraction and cardiac output were significantly improved （P 〈 0.05） in the cotransplantation group. Compared with the model group, Nkx2.5 and GATA-4 mRNA expression significantly increased in the BMSCs and cotransplantation group 4 weeks following transplantation. The increase in Nkx2.5 mRNA was significantly lower in the cotransplantation group than in the BMSCs group （P 〈 0.05）. CONCLUSION： EPCs preconditioned with Salvianolic acid B combined with BMSCs transplantation improved the expression of NKx2.5 and GATA-4, promoted cardiac function of acute myocardial infarction rat models.