骨髓间充质干细胞在梗死心肌移植再生后nesprin蛋白表达的实验研究

Study on the regeneration of transplanted mesenchymal stem cells in the infarcted myocardium and identify the expression of nesprin protein

目的:研究骨髓间充质干细胞(mesenchymal stem cells,MSCs)移植到急性心肌梗死区心肌样细胞再生后ne-sprin蛋白的表达情况。方法:大鼠16只,随机分为实验组和对照组,每组8只。利用密度梯度分离法和贴壁培养MSCs,流式细胞仪检测MSCs表面相关抗原、DAPI标记MSCs备用;SD大鼠开胸手术结扎心脏冠脉左前降支建立心肌梗死模型,并在实验组梗死区注射MSCs,对照组注射DMEM;术后3周取梗死区心肌,利用免疫细胞学化学方法鉴定肌节肌动蛋白和肌钙蛋白的表达,免疫荧光技术和蛋白质印迹鉴定nesprin蛋白在MSCs移植后的表达。结果:MSCs移植到梗死缺血区3周后可以用DAPI示踪、并有心肌细胞特异蛋白呈阳性表达;免疫荧光和蛋白质印迹鉴定在MSCs移植前后有nesprin蛋白的表达,移植后出现nesprin蛋白表达量的增加。结论:MSCs在梗死心肌内移植具有分化再生的能力,nesprin蛋白在MSCs移植后出现表达量的增加,可能在MSCs分化为心肌细胞样过程中起到一定的作用。

Objective：Identify the differentiation of rat bone mesenchymal stem cells（MSCs）into myocardium-like cells after MSCs was transplanted into acute myocardial infarction.Then,identify the expression of nesprin protein.Methods：Sixteen SD rats were randomly divided into experimental group （n=8） and the control group （n=8）.MSCs were isolated and purified from the bone marrow of rats by density gradient centrifugation and adhering to the culture plastics.Surface-associated antigens of MSCs were detected by flow cytometry.These cells were labeled with DAPI.SD rats were anesthetized,through thoracotomy,then ligated the left anterior descending coronary artery in order to establish model of myocardial infarction.The DAPI marking MSCs were injected into the infarcted area in the experimental group;DMEM was used in the control group.The expression of α-sarcomeric actin,troponin I were studied by immunocytochemistry three week later when MSCs were transplanted into acute myocardial infarction.The expression of nesprin protein in MSCs was identified by immunofluorescence and western-blotting before and after transplantation.Results：MSCs can be traced by DAPI three weeks later when MSCs were transplanted into the infarcted ischemic area.The expression of α-sarcomeric actin and troponin I were positive in MSCs three weeks after transplanting into the infarcted ischemic area.The expression of nesprin protein was positive in MSCs by immunofluorescence and western-blot whenever MSCs were transplanted or not.But,three weeks after MSCs were transplanted,the expression of nesprin protein in the infarcted ischemic area was higher than that of DMEM was used in the infarcted ischemic area.Conclusion：This study suggests that MSCs were capable to differentiation with subsequent myocardial regeneration,after being implanted into the infarcted myocardium;the expression quantity of nesprin protein was higher after MSCs were implanted.When MSCs are differentiating into cardiac myocyte-like,nesprin protein may play a role in this procession.