犬骨髓间叶干细胞体外定向诱导分化心肌细胞的实验研究  

Committed Differentiation of Mesenchymal Stem Cells From Canine Bone Marrow to Cardiomyocytes With 5-Azacytidine in Vitro

作　　者：任晓庆[1] 浦介麟[1] 张澍[1] 张明伟[2] 贾玉和[1] 吴国玫[1] 孟亮[1] 毛宁[2] 王方正[1] 

机构地区：[1]中国医学科学院中国协和医科大学心血管病研究所阜外心血管病医院心律失常诊治中心,北京市100037 [2]军事医学科学院细胞生物研究所

出　　处：《中国循环杂志》2004年第1期16-18,共3页Chinese Circulation Journal

基　　金：国家自然科学基金 (30 1 70 374);中国博士后科学基金 (2 0 0 2 0 32 1 2 8);教育部优秀教师基金资助课题

摘　　要：目的 :旨在建立骨髓间叶干细胞 (MSCs)体外定向诱导分化心肌细胞的方法 ,为心肌疾患的移值修复治疗提供成体干细胞来源。方法 :利用Percoll密度梯度法及MSCs黏附贴壁生长特性进行分离培养与扩增骨髓MSCs ,并予以鉴定证实。应用5 氮胞苷对培养早期的骨髓MSCs进行定向诱导分化心肌细胞。通过细胞形态学、细胞免疫组化、透射电镜等技术观察分化细胞的肌管 ,心肌细胞特异性蛋白与细胞特异性超微结构以鉴定诱导分化的效果。结果 :利用Percoll密度梯度法与细胞黏附贴壁生长特性 ,可分离培养与扩增足量骨髓MSCs。应用化学诱导剂 5 氮胞苷 10～ 2 0 μmol/L孵育早期培养的骨髓MSCs 4～ 5周 ,可见细胞形成肌管 ;肌细胞特异性蛋白α 肌动蛋白 ,肌球蛋白以及心肌细胞特异性分子标志肌钙蛋白I免疫组化染色阳性 ;透射电镜可见肌丝与房性颗粒。结论 :骨髓MSCs可在体外 5 氮胞苷的诱导下定向转化为具有典型结构的心肌细胞。Objective:To establish the method for committed differentiation of mesenchymal stem cells (MSCs) to cardiomyocytes in vitro,provide an ideal cell source for the restoration therapy of damaged or diseased cardiac tissue. Methods:Mesenchymal stem cells isolated from canine bone marrow with Percoll density gradient solution were culture-expanded because the cells attached and grew on dishes.MSCs were identified by surface antigenic features.In order to obtain committed differentiation of mesenchymal stem cells to cardiomyocytes,MSCs were cultured for 4～5 weeks under different concentrations of 5-azacytidine.The myotubes,cardiac specific antigens,cellular ultrastructure of differentiated cells were determined by morphology,immunohistochemical staining and transmission electron microscopg. Results:Mesenchymal stem cells were induced to differentiate to myotubes and myocyte-like cells by incubating the cells with 10～20 μmol/L 5-azacytidine.Myocyte antigens such as α-actin,α-actinin,myosin,and cardiomyocyte specific troponin I of differentiated cells were stained positively in immunohistochemistry.Transmission electron microscopg showed that differentiated cells with oval nuclei positioned in the central part of the cell had well-organized myofilaments and atrial granules. Conclusion:The mesenchymal stem cells from canine bone marrow could be differentiated into cardiomyocytes by 5-azacytidine in vitro.The differentiated cardiomyocytes would provide an ideal cell source for transplantation therapy of damaged cardiac muscle tissue.

关 键 词：骨髓 间叶干细胞 体外定向 诱导分化 心肌细胞 实验研究 

分 类 号：R542.2[医药卫生—心血管疾病]