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目的体外分离培养、扩增骨髓间充质干细胞(mesenchymal stem cells,MSCs),诱导其定向分化为软骨细胞,为MSCs应用于临床、构建组织工程气管软骨提供实验依据。方法采用全骨髓培养+贴壁纯化法分离培养MSCs,流式细胞仪鉴定MSCs,以转化生长因子β1(transforming growth factorβ1,TGF-β1)、胰岛素样生长因子1(insulin-like growth factor 1,IGF-1)作为主要的诱导因子诱导MSCs定向分化为软骨细胞。21 d后用Ⅱ型胶原免疫细胞化学染色法鉴定诱导出的细胞,在光学显微镜和电子显微镜下观察MSCs诱导前、后的细微和超微结构改变。结果诱导21 d后Ⅱ型胶原免疫细胞化学染色呈阳性。光学显微镜和电子显微镜检查显示:MSCs呈梭形,诱导后细胞增大,呈多边形、星形、三角形。透射电子显微镜显示:诱导后细胞器丰富,胞核增大,核仁增多。结论 MSCs诱导分化为软骨细胞的超微结构表现为细胞器丰富、胞核增大、核仁增多,提示细胞分化、代谢功能旺盛。
Objective To isolate and culture mesenchymal stem cells (MSCs) in vitro and induce them to differentiate into chondrocytes for the clinical application of MSCs in the construction of tissue engineering tracheal cartilage. Methods MSCs were isolated and cultured by whole bone marrow culture and adherent purification. MSCs were identified by flow cytometry. Transforming growth factor β1 (TGF-β1), insulin-like growth factor 1 -1) as the main inducing factor to induce MSCs to differentiate into chondrocytes. Twenty-one days later, the induced cells were identified by immunocytochemical staining of type II collagen and the ultrastructural changes before and after MSCs induction were observed under light microscope and electron microscope. Results The type Ⅱ collagen immunocytochemical staining was positive 21 days after induction. Light microscopy and electron microscopy showed that MSCs were spindle-shaped, and the cells were enlarged after induction. They were polygonal, star-shaped and triangular. Transmission electron microscopy showed that after induction, the organelles were rich, the nuclei were enlarged and the nucleoli were increased. Conclusion The ultrastructural differentiation of MSCs into chondrocytes is characterized by abundant organelles, enlarged nuclei and increased nucleoli, suggesting cell differentiation and strong metabolic function.