人脐带间充质干细胞移植对小型猪急性心肌梗死模型心肌细胞再生的影响

Effect of human umbilical cord mesenchymal stem cells transplantation on myocardial regeneration of mini-swine model with acute myocardial infarction

目的观察人脐带间充质干细胞移植治疗心肌梗死的可行性及机制,探讨经心外膜多点注射对小型猪急性心肌梗死模型心肌细胞再生的影响。方法无菌条件下采集足月儿的脐带,体外分离、培养、扩增人脐带间充质干细胞至第5代并以CM-Dil标记。取小型猪18例制作急性心肌梗死模型,并随机分为对照组、PBS组和移植组,每组6只。PBS组和移植组分别在梗死区注射PBS和脐带间充质干细胞。干预6周后行核素心肌灌注显像,观察梗死区灌注情况;通过免疫荧光法、Masson和TUNEL染色法观察梗死心肌中干细胞存活、增殖、分化情况和心肌纤维化、心肌细胞凋亡情况。结果与对照组及PBS组相比,移植组心肌灌注情况明显改善(P<0.01);移植后6周梗死区域可见移植的干细胞存活,其中部分向心肌和血管内皮细胞分化,亦可见固有心肌干细胞的募集与分化。TUNEL染色结果显示,移植组细胞凋亡明显减少(P<0.01)。Masson染色证实移植组的存活心肌明显增多而纤维组织明显减少(P<0.01)。结论人脐带间充质干细胞移植于心肌梗死区域后可部分存活并分化为心肌和血管组织,可促进固有心肌干细胞的募集和分化,减少细胞凋亡和组织纤维化,改善梗死区域的心肌存活情况,抑制心室重构,促进梗死心肌再生。

Objective To investigate the feasibility and mechanism of transplanted cells in the treatment of AMI and to detect the effects of epicardial multi-point injection in swine model of AMI on myocardial cell regeneration. Methods UC-MSCs were isolated from human umbilical cord in sterile conditions and expanded to the fifth generation. UC-MSCs were labeled with CM-Dil before transplantation. The 18 established AMI model miniswines were randomly divided into three groups, which were control group（n = 6）, PBS group（ n = 6） and transplantation group（ n = 6）. PBS and UC-MSCs were injected into infarction area in PBS group and transplantation group, respectively. Six weeks later, evaluation of perfu- sion in infarction area was performed by SPECT in each group. The animals were euthanized and the tissues in infarction ar- ea were harvested to analyze the survival, proliferation and differentiation of stem cells as well as to observe the myocardial fibrosis, apoptosis and ventricular reconstruction. Results The perfusion was significantly improved in the transplantation group as compared with the control and PBS groups（ P 〈 0.01 ）. Immunofluorescence results confirmed that the transplanted UC-MSCs were still alive and part of them appeared to have differentiated into cardiomyocytes and vascular endothelium cells six weeks after transplantation. Meantime, it was also observed that resident CSCs differentiated into neonatal cardio- myocytes and vascular endotheliums. The TUNEL analysis indicated that the number of apoptosis was significantly reduced in the transplantation groups（P 〈0.01 ）. Masson staining confirmed that there were more viable myocardium and less fi- brous tissue in the transplantation group （P 〈 0.01 ）. Conclusions Transplantion of human UC-MSCs into infarction area of AMI was suit for the survival and differentiation of myocardial cell, as well as the promotion of CSCs recruitment and dif- ferentiation. In addition, UC-MSCs transplantation reduced myocardial cell apoptosis and fibrosis, enhanced viable myocar- dium and thus suppressed the ventricular reconstruction.