摘要
目的采用骨髓间充质干细胞(MSCs)体外诱导为心肌样细胞,为干细胞移植治疗心衰提供一种新的细胞材料。方法取SD大鼠四肢骨骨髓,分离培养MSCs,分别用终浓度为5、10、15、20μmol/L的5-氮胞苷(5-aza)定向诱导,相差显微镜观察细胞形态学变化,应用免疫细胞化学、激光扫描共焦显微镜技术检测结蛋白(desmin),α-横纹肌肌动蛋白(α-sarcomeric actin)、心肌特异性肌钙蛋白(C-TnT)的表达,透射电镜鉴定。在诱导后7d、21d和28d,3个时间点以半定量RT-PCR方法检测细胞心肌早期转录因子GATA4和心肌特异性肌凝蛋白重链αMHC的表达。结果MSCs体外经5-aza诱导后分化的细胞desmin、α-sarcomericactin、C-TnT均表达阳性,5μmol/L组阳性表达较高,透射电镜下可见到平行排列的肌丝。RT-PCR结果显示,GATA4于诱导后7d弱表达,21d表达增强,28d表达减弱。αMHC在诱导后7d不表达,2ld弱表达,28d表达明显。结论骨髓间充质干细胞能够在体外被诱导分化为心肌样细胞,是自体心肌细胞一种良好的供体来源。
Objective To establish a new cell resource of stem cell transplantation for heart failure therapy, by using cardiomyocytes from rat marrow mesenchymal stem cells（MSCs）. Methods MSCs were isolated and cultured from rat bone marrow, then induced by 5-aza （5, 10, 15, 20 μmol/L） for 24 hour. The cultured ceils were observed with phase-contrast microscopy. The immunohistochemical technique and laser scanning confocal microscopy （LSCM） were applied for revealing the expressions of desmin, α-sarcometic aetin and C-TnT. The induced cells were evaluated by a transmission electron microscope. GATA4 and α-MHC expressions were detected by relative quantitative RT-PCR after 7, 21, 28 days of inducement respectively. Results MSCs induced in vitro by 5-aza could be identified by the positive staining for desmin, α-sarcomeric actin and C-TnT. The number of cells stained positively in the 5 μmol/L was greater than that of other groups. Transmission electron microscope showed that paralleled myofilaments were formed. RT-PCR assessment showed that the differentiated cells began to express GATA4 from day 7 to day 28 of differentiation and began to express αMHC from day 21 to day 28 of differentiation. Conclusion Cardiomyocyte-like cells can be generated from MSCs in vitro and the marrow mesenehymal stem cells are one of the best resources of donor cardiomyocytes.
出处
《解剖学报》
CAS
CSCD
北大核心
2007年第1期70-74,共5页
Acta Anatomica Sinica
关键词
骨髓间充质干细胞
5-氮胞苷
心肌细胞
激光扫描共焦显微镜
大鼠
Bone marrow mesenehymal stem cells
5-azaeytidine
Cardiomyocyte-like cells
Cell differentiation
Lasers canning eonfocal microscopy
Rat
