贴壁法分离培养大鼠骨髓间充质干细胞的生物学特性

THE BIOLOGICAL CHARACTERISTICS OF RAT BONE MARROW-DERIVED MESENCHYMAL STEM CELLS ISOLATED AND CULTURED WITH ATTACHMENT METHOD

目的建立一种简便有效的体外分离纯化及培养扩增大鼠骨髓间充质干细胞(MSCs)的方法。研究MSCs的生物学特性,为血管组织工程提供理想的种子细胞。方法贴壁培养法分离纯化大鼠MSCs体外培养和连续传代,在倒置显微镜下连续观察细胞的形态变化;利用MTT法测定MSCs的生长曲线;行免疫组化方法鉴定MSCs膜抗原;分别加成骨、成脂肪诱导剂后MSCs体外培养1到3周,分别做碱性磷酸酶(ALP)、VonKossa染色及油红O染色,观察细胞形态变化、成骨及成脂肪分化结果。结果MSCs体外培养生长状况良好,呈均一的成纤维细胞样,表达波形蛋白(Vimentin)、α-平滑肌肌动蛋白(α-SMA),不表达层粘连蛋白(Laminin)、CD34、VIII因子相关抗原(VIII)。经体外诱导后具有多向分化潜能。结论贴壁培养法能有效分离纯化大鼠MSCs,用此方法培养的细胞生长稳定,增殖能力活跃,具有MSCs的一般生物学特性,为其成为血管组织工程理想的种子细胞提供了进一步的支持。

Objective To establish a simple and effective method of isolation, purification and culture of rat bone marrow-derived mesenchymal stem cells in vitro, and explore their biological characteristics in order to provide an ideal seed cell for vascular tissue engineering Methods MSCs were isolated and purified by the attachment culture method. The cell growth was measured by MTT method, and membrane antigens were detected with immunohistochemistry under an inverted microscope. Osteoblastic differentiation and adipocytic differentiation were induced and observed by culturing confluent MSCs from 1 to 3 weeks in medium supplemented with different inducers. Results The morphology of MSCs was identical fibroblast-like and the cells showed active proliferative ability. Vimentin and α-SMA antigen could be detected, but not laminin, CD34 and Ⅷ antigen. 3 weeks after in the osteoblastic inductive medium, the treated population contained numerous alkaline phosphatase positive cells and von Kossa positive extracellular matrix. 2 weeks after in adipocytic inductive medium, lipid accumulation was clearly visible and stained with 0 Red Oil. Conclusion The attachment culture method can be used to isolate and purify MSCs. Cultured MSCs show stable and rapid growth in vitro and have general biological characteristics, which further make them as ideal seed cells for vascular tissue engineering.