人脐带干细胞移植在心肌梗死大鼠心肌内的迁移、分化及对心脏功能的影响

目的 探讨脐带干细胞移植对大鼠心肌梗死后心脏功能的影响及细胞在心肌内的迁移和分化。方法 2010年10月—2011年6月收集南京鼓楼医院妇产科10例健康胎儿脐带,采用胶原酶胰酶消化法分离脐带干细胞。选取8周龄、体质量260～280 g 的SD雄性大鼠30只,采用结扎大鼠冠状动脉前降支建立心肌梗死模型。造模后第14天行心脏超声检查,将造模成功的大鼠随机分为移植组(12只)和对照组(11只),并分别经心肌每只大鼠注射200 μl人脐带干细胞悬液和PBS。在细胞移植前和移植后第14、28天行心脏超声心动图检查,测定心脏功能。在移植后28天,处死移植组大鼠,制作心脏冷冻切片。切片采用血管特异性平滑肌动蛋白α(α-SMA)抗体、内皮特异性血管性血友病因子(vWF) 抗体及心肌特异性肌钙蛋白T (cTnT)抗体染色,在荧光显微镜下观察脐带干细胞的存活、迁移情况,以及血管平滑肌细胞、血管内皮细胞和心肌细胞的分化情况。结果 30只大鼠中造模成功23只,脐带干细胞移植后存活20只,其中移植组10只,对照组10只。超声心动检查,与移植前比较,两组大鼠干细胞移植后第14、28天的左室射血分数、短轴缩短率均有明显增加,差异均有统计学意义(t_射血分数=3.864、3.690,t_{短轴缩短率}=6.397、5.904,P值均<0.05)。移植后组间比较:干细胞移植后第14天,两组大鼠的左心室射血分数差异无统计学意义(P＞0.05),而左室短轴缩短率移植组大于对照组,差异有统计学意义(t=2.771,P<0.05);干细胞移植后第28天,移植组大鼠的左室射血分数和短轴缩短率均明显高于对照组,差异均有统计学意义(t=2.977、2.140,P值均<0.05)。荧光显微镜下观察,移植组可见脐带干细胞能够在梗死心肌内存活并发生迁移,α-SMA抗体、vWF抗体和cTnT抗体染色显示移植的脐带干细胞能够分化为血管平滑肌细胞、内皮细胞和心肌细胞。结论 脐带干细胞能够在梗死心肌内迁移,在局部梗死心肌微环境下,能够分化为血管平滑肌细胞、内皮细胞和心肌细胞,促进血管新生和心肌再生,从而促进心脏功能的恢复。

Cell engraftment,differentiation and contribution to cardiac function recovery of umbilical cord derived mesenchymal stem cells

Objective To investigate the survival and engraftment of human umbilical cord derived mesenchymal stem cells (UC-MSCs) and its contribution to cardiac functional recovery in a rat myocardial infarction(MI) model.Methods From October 2010 to June 2011, 10 human umbilical cord tissues were obtained from Obstetrics and Gynecology Department, Nanjing Gulou Hospital. The UC-MSCs were isolated by enzyme digestion. Thirty old male SD rats (8-week, weight 260-280 g) were used to establish MI model by high ligation of left anterior descending coronary artery. Fourteen days after induction of MI, echocardiography was used to assess the cardiac function, and the successfully established MI rats were randomly divided into cell transplantation group and phosphate buffered saline (PBS) control group. Cardiac function was assessed by echocardiography pre-transplantation, 14 and 28 days after cell transplantation. At day 30, the rats were sacrificed to make heart tissue frozen sections. Vascular smooth muscle actin (α-SMA), von Willebrand factor (vWF), and cardial troponin-T (cTnT) staining were performed and immunofluorescence was used to investigate the survival, engraftment and differentiation of transplanted cells.Results Twenty-three MI rats were successfully established in 30 rats, and 20 rats survived after cell transplantation, 10 in transplantation group and 10 in control group. By echocardiography, compared with pre-transplantation, the improvement of left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were observed, and the differences were statistically significant (t_LVEF=3.864, 3.690, t_LVFS=6.397, 5.904, all P values<0.05).Group comparison: at day 14, there was no difference in LVEF (P＞0.05) but LVFS was significantly higher in transplantation group (t=2.771, P<0.05). At day 28, both LVEF and LVFS were better in transplantation group (t=2.977, 2.140, all P values<0.05). Immunofluorescence revealed that the transplanted UC-MSCs survived and engrafted in the infarted myocardium and α-SMA, vWF, and cTnT staining showed that transplanted UC-MSCs differentiated into smooth muscle cells, endothelial cells, and cardiomyocytes.Conclusions UC-MSCs can engraft the infarcted myocardium and have the ability to differentiate into smooth muscle, endothelial, and cardiomyocytic cells in the local MI environment, contribute to myocardial regeneration, angiogenesis and the cardiac functional recovery.