经5-氮胞苷诱导的骨髓间充质干细胞移植治疗大鼠急性心肌梗死的实验研究

Treatment of rat acute myocardial infarction with transplantation of BMMSCs induced by 5-azacytidine

目的观察经5-氮胞苷(5-Aza)能否诱导骨髓间充质干细胞(BMMSCs)分化成类心肌细胞,并移植入急性心肌梗死组织,观察对大鼠心功能的影响。方法体外分离培养、鉴定骨髓间充质干细胞,经5-Aza诱导,鉴定其表达心肌细胞特有蛋白,体外DAPI标记。SD大鼠随机分为分3组,对照组:于冠状动脉LAD结扎建立急性心肌梗死模型后1 h在梗死周边区用微量注射器分4点注射生理盐水;5-Aza组:造模后于相同部位注射等量经5-Aza诱导的BMMSCs;BMMSCs组:造模后于相同部位注射等量未诱导的BMMSCs。术后3 h,3 d,4 w追踪被移植细胞是否存活于心肌组织中,术后4 w通过测定大鼠左室收缩压、舒张末压、压力变化最大上升与最大下降速率以评价心功能的改变。结果 (1)经5-Aza诱导后,部分BMMSCs形态发生变化,并开始表达β-肌球蛋白重链、肌钙蛋白T等心肌细胞特有蛋白;(2)荧光显微镜观察发现,术后3 h,3 d,4 w,在5-Aza组及BMMSCs组均有DAPI标记的BMMSCs存活在梗死心肌组织中;(3)与BMMSCs组比较,5-Aza组左室收缩压升高(P<0.05),舒张末压明显降低(P<0.05)、左心室内压最大上升和下降速率显著增快(P<0.05)。结论 BMMSCs经5-Aza诱导后可向心肌细胞分化,分化后的类心肌细胞可用来治疗急性心肌梗死(AMI)。

Objective To investigate whether 5-azacytidine（5-Aza） induces bone marrow mesenchymal stem cells（BMMSCs） to differentiate into cardiomyocyte-like cells（CMCs）,and the effects of CMCs transplantation on cardiac function in rabbits after acute myocardial infarction（AMI）.Methods BMMSCs were purified,cultured and identified in vitro,and then were induced by 5-Aza.Before transplantation,the induced BMMSCs were labeled with DAPI at 4 weeks following induction.Sprague-Dawley（SD） rats were randomly assigned into three groups.Left anterior descending artery（LAD） was ligated to establish a rat model of AMI.In control group,saline was infused into the surrounding infarct area at 4 points using a microsyringe at 1 hour following model induction by coronary artery ligation.In 5-Aza group,an equal volume of BMMSCs induced by 5-Aza was injected at the same region.In BMMSCs group,an equal volume of BMMSCs was injected at the same region following model establishment.A fluorescence microscope was used to observe whether BMMSCs survived in the infarcted myocardial tissue at 3 h,3 d or 4 w following transplantation,and the hemodynamic testing was used to evaluate cardiac function.Results After induced by 5-Aza,part of BMMSCs began to express the specific protein of myocardical cell,just as beta myosin heavy chain（β-MHC）,troponin T,and so on.Fluorescence microscopy results demonstrated that DAPI-labeled BMMSCs were survived in the myocardium of 5-Aza group and BMMSCs group.Compared with BMMSCs group,the survival rate of DAPI-labeled transplanted cells was increased,and the cardiac function improved in 5-Aza group.Conclusion It is confirmed that BMMSCs can be differentiated into CMCs by induction with 5-Aza,and the CMCs can be used to improve the cardiac function of AMI rats.