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目的探讨碱性成纤维细胞生长因子(bFGF)体外诱导骨髓间充质干细胞(BMSCs)分化为心肌细胞的可行性。方法取SD大鼠四肢骨骨髓,分离培养BMSCs,应用bFGF对其定向诱导,倒置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-actin)、心肌特异性肌钙蛋白(C-TnT)的表达,透射电镜观察分化细胞的超微结构变化。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α肌球蛋白重链(α-MHC)的表达。结果原代细胞培养48 h后,大部分细胞呈梭形成纤维细胞状,并带有2～3个长的突起,小部分细胞呈扁平形。给予bFGF诱导后,细胞形态发生明显变化,诱导1周的BMSCs体积增大呈肌细胞样短柱状或长梭形,平行排列生长,诱导4周的BMSCs细胞多呈短柱状,相邻细胞紧密接触形成类肌管样结构,排列具有明显的方向性。经bFGF诱导后分化的细胞胞质内desmin、α-actin和C-TnT染色均为阳性,其中α-actin、desmin阳性率较高分别为58.64%和33.82%,C-TnT阳性率较低为28.94%。激光扫描共焦显微镜观察可见,细胞质内desmin和α-actin的表达呈红色,C-TnT的表达呈绿色,当前两者分别与C-TnT同时被观察时均可见重叠的部位变成黄色。透射电镜观察到分化细胞呈杆状,胞核卵圆形,位于细胞中央,胞质中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,GATA-4于诱导后7d弱表达,21d表达增强,28d表达减弱。心肌特异性α肌球蛋白重链在诱导后7d不表达,21d弱表达,28d表达明显。结论 bFGF可以诱导BMSCs定向心肌分化,是有效的心肌细胞分化诱导剂。
Objective To investigate the feasibility of basic fibroblast growth factor (bFGF) inducing bone marrow mesenchymal stem cells (BMSCs) to differentiate into cardiomyocytes in vitro. Methods BMSCs were isolated from bone of extremities of SD rats. BMSCs were isolated and cultured. The cells were induced by bFGF. The morphological changes of cells were observed by inverted phase contrast microscope. Immunocytochemistry and laser scanning confocal microscopy were used to detect the expression of desmin, The expression of α-actin and cardiac troponin (C-TnT) was observed by transmission electron microscopy. The ultrastructural changes of differentiated cells were observed by transmission electron microscopy. The expression of GATA4 and α-MHC in myocardial cells were detected by semi-quantitative RT-PCR at 7d, 21d and 28d after induction. RESULTS: After cultured for 48 h, most of the cells were fibroblast-like with 2-3 protuberances and a small number of cells were flattened. After induction of bFGF, the morphology of cells changed significantly. The volume of BMSCs induced by bFGF increased in a short columnar or long spindle shape in parallel. The number of BMSCs induced by bFGF was mostly short columnar and the adjacent cells were in close contact The formation of myotubes-like structure, arranged in a clear direction. The expression of desmin, α-actin and C-TnT in the differentiated cells induced by bFGF were all positive, the positive rates of α-actin and desmin were 58.64% and 33.82% respectively, and the positive rate of C-TnT was low 28.94%. Laser scanning confocal microscopy showed that the expression of desmin and α-actin in the cytoplasm was red, and the expression of C-TnT was green. At the same time, both of them were observed to be yellow with the overlap of the C-TnT. Transmission electron microscopy showed that the differentiated cells were rod-shaped, oval nucleus, located in the center of the cells, the cytoplasm can be seen in parallel with the arrangement of myofilaments, a large number of rough endoplasmic reticulum and mitochondria, rich in glycogen and ribosomes. RT-PCR results showed that GATA-4 was weakly expressed on day 7 and increased on day 21, and decreased on day 28. Myocardial specific α-myosin heavy chain did not express at 7d, weakly expressed at 21d, and obviously expressed at 28d. Conclusion bFGF can induce BMSCs to differentiate into cardiomyocytes and is an effective cardiomyocyte differentiation inducer.