QY1骨髓多能间质干细胞系多向分化潜能的检测

Pluripotential differentiation of QY1 bone marrow mesenchymal stem cell line

目的:检测QY1骨髓间质干细胞系在体外向脂肪细胞、软骨细胞、成骨细胞、心肌细胞、血管内皮细胞和神经细胞分化的能力。方法:选用第5代QY1骨髓间质干细胞系,分别用成脂培养基、成软骨培养基、成骨培养基、5?氮胞苷、血管内皮生长因子、神经细胞培养液(诱导液1为10mmol/Lβ-巯基乙醇;诱导液2为2%二甲基亚砜和10-8mol/L地塞米松)对其进行定向诱导分化。采用染色法、免疫组化法、RT-PCR法鉴定分化后的细胞。结果:在成脂培养基培养21d左右出现大量含脂滴的脂肪细胞,苏丹III染色法检测脂肪细胞胞浆呈桔红色。在成软骨培养基培养21d左右有软骨结节形成,阿尔新蓝染色法将软骨结节染为深蓝色。在成骨培养基培养35d左右有凸出的骨结节形成,VonKossa染色法显示有黑色矿化结节形成。在10μmol/L5?氮胞苷诱导液中能出现自发性搏动的心肌细胞和肌管样结构,免疫组化法显示心肌特异性的表面抗体α-横纹肌肌动蛋白、心肌连接蛋白-43表达阳性,RT-PCR法检测有心肌特异性因子α-肌球蛋白重链表达。在含有50ng/mL血管内皮生长因子的诱导液中能出现呈直线排列的血管内皮细胞和血管内皮细胞网状结构,免疫组化法显示血管内皮特异性表面抗体CD31和VIII因子表达阳性。神经诱导液1处理组出现神经元,免疫组化法显示胶质纤维酸性蛋白表达阴性,神经元特异性核蛋白表达阳性;神经诱导液2处理组出现神经胶质细胞,免疫组化法显示胶质纤维酸性蛋白表达阳性,神经元特异性核蛋白表达阴性。结论:本研究证实QY1骨髓间质干细胞系细胞具有分化为脂肪细胞、软骨细胞、成骨细胞、心肌细胞、血管内皮细胞、神经元和神经胶质细胞的潜能,提示QY1骨髓间质干细胞系具有向多个胚层、7个方向分化的多向潜能。

Objective To explore the ability of QY1 bone marrow mesenchymal stem cell （ MSCs ） line cells to differentiate into adipocytes, chondrocytes, osteoblasts, cardiacmyocytes, vascular endothelial cells, and neural cells in vitro. Methods The QY 1 cells at passage 5 were treated with the adipogenic medium, the chondrogenic medium and the osteogenic medium, 5-azacytidine, vascular endothelial growth factor and neural cell medium（ revulsant 1 was 10 mmol/L β-mercapto-ethanol ; revulsant 2 was 2 % dimethylsulfoxide and 10^-8 mol/L dexamethasone ） in culture respectively in vitro. The differentiated cells were identified by staining, immunohistochemistry and RT-PCR. Results The differentiated cells induced by the adipogenic medium formed adipocytes and contained fat lipid droplets, which were stained positively with Sudan Ⅲ after 21 days of culture. The differentiated cells induced by the chondrogenic medium formed chondrogenic nodules, which were stained positively by Alcian blue at pH 1.0 after 21 days of culture. The differentiated cells induced by the osteogenic medium formed osteogenic nodules, which were stained positively by Von Kossa staining after 35 days of culture, and the secretion of a calcified extracellular matrix as black nodules was observed. The differentiated cells treated with 10 μmol/L 5-azacytidine could beat spontaneously and formed myotube structures, which were identified by the positive immunohistochemistry staining with anti-α-sarcomeric antibody and anti-Cx-43 antibody. The expression of α-myosin heavy chain was also observed by RT-PCR. The differentiated cells treated with 50 ng/mL vascular endothelial growth factor could form vascular endothelial cells and vascular endothelial web like structure, which were identified by the positive immunohistochemistry staining with CD31 and Factor Ⅷ. The differentiated cells induced by revulsant 1 were positive in the immunohistochemistry staining with neuron-specific nuclear protein, while the expression of glial fibrillary acidic protein was negtive. The differentiated cells induced by revulsant 2 were positive in the immunohistochemistry staining with glial fibrillary acidic protein, while the expression of neuron-specific nuclear protein was negative. Conclusion QY 1 bone marrow mesenchymal stem cell line has the ability to differentiate into adipocytes, chondro- cytes, osteocytes, cardiomyocytes, vascular endothelial cells, neurons and neural glial cells in vitro. A bone marrow mesenchymal stem cell line cell can at least differentiate into 7 types of cells, which come from mesoderm and ectoderm.