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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。
OBJECTIVE: To induce bone marrow mesenchymal stem cells (MSCs) to differentiate into cardiomyocyte-like cells by bone morphogenetic protein-2 (BMP-2) in vitro and explore the induction of MSCs to cardiomyocytes. METHODS: Bone marrow was isolated from the extremities of SD rats. MSCs were isolated and cultured. The morphological changes of the cells were observed by BMP-2 and phase contrast microscopy. Immunocytochemistry and laser scanning confocal microscopy were used to detect the expression of desmin, Α-sarcomeric actin, cardiac-specific troponin (C-TnT) were detected by transmission electron microscopy. The expressions of GATA4 and α-MHC were detected by semi-quantitative RT-PCR at 7d, 21d and 28d after induction. Results After BMP-2 induced MSCs cells extended pseudopodia, the direction of alignment became more and more consistent. The positive rate of α-striated muscle actin and C-TnT of MSCs differentiated by BMP-2 in vitro was high, the positive rate of desmin and α-transverse actin was 37.28% and 63.94 %, While the low positive rate of C-TnT was 34.66%. Transmission electron microscopy can be seen in parallel arranged myofilament, a large number of rough endoplasmic reticulum and mitochondria, rich in glycogen and ribosomes. RT-PCR results showed that GATA4 was weakly expressed on day 7 and increased on day 21, and decreased on day 28. α-MHC was not expressed on the 7th day after induction, but weakly on the 21st day, and significant on the 28th day. Conclusion Bone marrow mesenchymal stem cells can be induced to differentiate into cardiomyocyte-like cells under the induction of BMP-2, which is a good donor source of autologous cardiomyocytes.