论文部分内容阅读
目的探讨磁共振(MRI)下活体动态示踪小型猪骨髓间充质干细胞(MSCs)经冠脉移植治疗心肌梗死的可行性及移植后8周MSCs的转归。方法采用密度梯度离心法获取小型猪自体骨髓MSCs,用携带绿色荧光蛋白(GFP)基因的慢病毒载体转染MSCs,并用超顺磁性氧化铁(SPIO)体外标记MSCs-GFP。采用经皮球囊封堵法制备急性心肌梗死(AMI)模型,随机分为2组,MSCs移植组(n=8),经冠脉移植双标记的MSCs到小型猪心肌梗死区域,对照组(n=5),移植等量生理盐水,移植后24 h、3周、8周在MRI下进行活体动态示踪。8周后离体组织学观察大体形态学改变及移植细胞存留情况。结果普鲁士蓝染色显示25μg Fe/mL SPIO与0.8μl/mL阳离子脂质体联合对MSCs的标记率达95%～100%,标记后细胞活力和增值能力较前改变无统计学差异(P>0.05),对GFP的表达无影响,双标记细胞仍具备成骨、成脂及成心肌分化能力,心肌细胞表面标记物α-MHC和actinin表达阳性。双标记MSCs经冠脉移植后24 h,3周,8周MRI成像均可见梗死区域的室间隔有明显区别于周边组织的低信号,信号强度(SI)改变分别为52.98%±10.74%,21.53%±5.40%,6.23%±2.01%(P<0.05),对照组无可见低信号区域。组织学检查示MSCs移植组心梗瘢痕面积明显缩小(P<0.05),HE染色见心梗区域心内膜下出现再生心肌,普鲁士蓝染色可见有蓝色颗粒聚集,荧光显微镜下同一切片GFP表达阳性,CD68(单核巨噬细胞表面标记物)表达阴性。每高倍镜视野下心梗边缘区和梗死区的蓝色颗粒数分别为36.2±3.8和9.7±2.1(P<0.05)。对照组普鲁士蓝染色阴性。结论 GFP/SPIO双标记MSCs是安全的,且不改变干细胞的生物学特性。经冠脉移植双标记MSCs可在MRI成像下表现出理想的信号强度,示踪时间长达8周,MSCs移植可减小心梗瘢痕面积,促进心肌再生,8周后SPIO主要定居于心梗边缘区,仍存在于MSCs中,SPIO活体动态示踪小型猪骨髓MSCs移植治疗心肌梗死是可行的。
Objective To investigate the feasibility of using live dynamic tracing of mini-pig bone marrow mesenchymal stem cells (MSCs) via coronary artery transplantation under magnetic resonance imaging (MRI) in the treatment of myocardial infarction and the outcomes of MSCs 8 weeks after transplantation. Methods Autologous bone marrow MSCs were obtained by density gradient centrifugation. MSCs were transfected with lentivirus vector carrying green fluorescent protein (GFP) gene and labeled with MSCs-GFP by superparamagnetism iron oxide (SPIO). Acute myocardial infarction (AMI) model was established by percutaneous balloon occlusion. The rats were randomly divided into 2 groups: MSCs transplanted group (n = 8), double labeled MSCs were transplanted into mini-swine myocardium, and the control group n = 5). The rats were transplanted with the same volume of saline, and were dynamically tracked by MRI at 24 h, 3 weeks and 8 weeks after transplantation. After 8 weeks, the morphological changes and the survival of transplanted cells were observed in vitro. Results Prussian blue staining showed that the labeled rate of MSCs was 95% ~ 100% with the combination of 25μg Fe / mL SPIO and 0.8μl / mL cationic liposome. There was no significant difference in cell viability and proliferation after labeling (P> 0.05 ), Had no effect on the expression of GFP. Double labeled cells still had osteogenic, adipogenic and cardiomyogenic potential, and positive expression of α-MHC and actinin on myocardial cell surface markers. Dual labeled MSCs showed low signal intensity in peripheral ventricular septum with MRI at 24 h, 3 wk and 8 wk after coronary transplantation. The signal intensity (SI) changes were 52.98% ± 10.74% and 21.53 % ± 5.40% and 6.23% ± 2.01%, respectively (P <0.05). There was no low signal area in the control group. Histological examination showed that the area of myocardial infarction in MSCs transplantation group was significantly reduced (P <0.05). HE staining showed myocardial regeneration under the endocardium in myocardial infarction area. Blue particles were observed in Prussian blue staining. GFP expression in the same section under fluorescence microscope Positive, CD68 (monocyte macrophage surface marker) expression was negative. The number of blue particles in the marginal zone and the infarct zone of MI in high magnification were 36.2 ± 3.8 and 9.7 ± 2.1 (P <0.05) respectively. Prussian blue staining in the control group was negative. Conclusion GFP / SPIO double labeled MSCs are safe and do not change the biological characteristics of stem cells. The double labeled MSCs by coronary artery can show the ideal signal intensity under MRI imaging, the tracing time is up to 8 weeks. MSCs transplantation can reduce the area of myocardial infarction scar and promote myocardial regeneration. After 8 weeks, SPIO mainly settles in myocardial infarction Marginal zone, still exists in MSCs, SPIO in vivo dynamic tracing of mini-pig bone marrow MSCs transplantation is feasible for myocardial infarction.