【摘要】 目的应用骨形态蛋白2(BMP-2)体外诱导骨髓间充质干细胞(MSCs)向心肌样细胞分化,探索MSCs向心肌细胞分化的诱导方法。方法取SD大鼠四肢骨骨髓,分离培养MSCs,应用BMP-2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28d3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α-肌凝蛋白重链(α-MHC)的表达。结果BMP-2诱导后的MSCs细胞伸出伪足,排列方向渐趋一致。MSCs体外经BMP-2诱导后分化的细胞结蛋白、α-横纹肌肌动蛋白、C-TnT均表达阳性,结蛋白、α-横级肌肌动蛋白阳性率较高,分别为37.28%和63.94%,而C-TnT阳性率较低为34.66%。透射电镜下可见到平行排列的肌丝,大量的粗面内质网和线粒体,富含糖原和核糖体。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。α-MHC在诱导后7d不表达,21d弱表达,28d表达明显。结论骨髓间充质干细胞在BMP-2诱导下可定向分化为心肌样细胞,是自体心肌细胞的一种良好供体来源。更多还原

【Abstract】 Objective To explore the optimal condition for the differentiation of rat bone marrow mesenchymal stem cells(MSCs) into cardiomyocyte-like cells in vitro by using bone morphogenetic protein 2(BMP-2). Methods SD rat MSCs were isolated from rat bone marrow and cultured,then induced by BMP-2 for 72 hours. The cultured cells were observed by phase-contrast microscope. The immunohistochemical technique and laser scanning confocal microscope (LSCM) were used for detecting of the expression of desmin,α-sarcomeric actin and C-TnT. The induced cells were evaluated by a transmission electron microscope. GATA4 and α-MHC expression were detected by relative quantitative RT-PCR after 7,21,28 days of induction respectively. Results MSCs induced by BMP-2 stretched out pseudopodium.The direction of the cell arraying was similar gradually. MSCs induced by BMP-2 could be identified by the positive staining for desmin,α-sarcomeric actin and C-TnT. Desmin、α-sarcomeric actin-positive cells made up higher of all MSCs than C-TnT-positive cells. Desmin and α-sarcomeric actin-positive cells were about 37.28% and 63.94%. C-TnT-positive cells were about 34.66%. Transmission electron microscope showed that the induced cells had a cardiomyocyte-like ultrastructure:the nuclei were positioned in the center of the cell. A lot of mitochondria,rough endoplasmic reticulum and ribosome were founded in plasm,and paralleled myofilaments could be seen in cytoplasma or beside the adge of membrane. 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 MSCs can differentiate into cardiomyocytes in vitro after induced by BMP-2,which suggests that MSCs might be one of the best resources of donor cardiomyocytes.更多还原