骨髓间充质干细胞诱导分化及其移植对兔心肌梗死后心功能的影响

Cellular Cardiomyoplasty with Myogenic Differentiation of BoneMarrow Mesenchymal Stem Cells Improves Cardiac PerformanceInduced by Infarct in Rabbits

目的采用骨髓间充质干细胞(MSCs)体外转化为肌细胞,为心力衰竭的细胞心肌成形提供较为理想的移植细胞。方法从兔股骨头处抽取骨髓,用Ficoll细胞分离液分离MSCs,体外培养,经5-氮杂胞甙(5aza)刺激24h后,用免疫组织化学方法检测肌源细胞中肌节肌动蛋白(Actin)、肌钙蛋白Ⅰ(Troponin Ⅰ);用RT-PCR检测肌球蛋白重链(MHC)的基因表达;用电镜观察肌源细胞的超微结构。并将诱导分化后的MSCs移植到结扎兔冠状动脉前降支致心肌梗死(MI)的模型中。移植后6周用心脏超声多普勒和血流动力学参数评价心功能。结果MSCs经5-aza刺激后,免疫组化染色肌节肌动蛋白、肌钙蛋白Ⅰ呈阳性;有肌球蛋白重链的基因表达;有多核肌管形成;透射电镜下可见明显的肌丝形成、糖原颗粒丰富、核居中。诱导分化MSCs移植组的左室射血分数、二尖瓣血流E／A左室前壁和间隔收缩速度均有显著增加(P＜0.05),左室舒张末期压有显著下降(P＜0.05)。结论MSCs可在体外环境下向肌细胞转化,移植诱导分化的MSCs至MI区域后,能改善MI后的心功能。

Objective To culture rabbit bone marrow mesenchymal stem cells(MSCs) for inducing them into myogenic cells in vitro. To test hypotheis that the induced cells can improve cardiac function in postinfarcted rabbits by using the induced cells autotransplanted into infracted myocardial tissue. Methods New Zealand White Rabbits were used as tested animals. In vitro studies: Isolation and primary culture of MSCs from the femoral bones of donor rabbits were performed according to Wakitani' s method. Then MSCs were cultured in cell culture medium with 5-azacytidine(5-aza, 8 μmol/L)for 24 hours. The myogenic characteristics of the induced cells were examined. In vivo studies: A myocardial infarction was created in 40 rabbits by coronary ligation. They were divided into 4 groups. Animals in the three groups were intramyocardially injected 2 weeks after the infarction with 5-aza-induced MSCs (group 1, n=7), cultured MSCs (group 2, n=6) or free-serum culture medium (group 3, n=7), Chest was just opened in group 4. Left ventricular function was assessed by echocardiography 8 weeks after MI. A fluid-filled catheter was introduced into the carotid and left ventricular end-diastolic pressure (LVEDP) was analyzed. Results MSCs cultured with 5-aza formed myotubules and results ofimmunohisto-chemically staining of actin, troponin I was positive. The induced cells had expression of myosin heavy chain. Transmission electron microscopy revealed a cardiomyocyte-like ultrastructure, including myofilaments, a centrally positioned nucleus, and rich glycogen granules. At the end of 8-weeks study, left ventricular ejection fraction(LVEF)was increased in group 1 compared with that in group 2 and 3(P<0.05) . Myocardial systolic velocity of anterior and anteroseptal wall significantly increased in group 1. LVEDP significantly decreased in group 1. Conclusion MSCs cultured with 5-aza (8 μmol/L) differentiate into cardiac-like muscle cells in vitro and in vivo in ventricular scar tissue and improve myocardial function in postinfarcted rabbits.