【摘要】 目的评估应用SD乳鼠骨髓的改良筛选法分离培养骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSCs)的可行性及优势,探讨一种简单高效的BMSCs体外分离、纯化及扩增的方法。方法选择7d龄SD大鼠6只,无菌解剖大鼠双侧股骨,于完全培养基浸泡、剪碎,反复吹打暴露骨髓腔,高速震荡3 min后取上清液接种于培养瓶中,24 h后半量换液,48 h后全量换液,以后隔天换液。应用CCK-8法测定生长曲线;成骨分化诱导3周后进行茜素红染色,成脂分化诱导4周后进行油红"O"染色;应用流式细胞仪鉴定细胞表面抗原。结果原代培养的BMSCs细胞生长曲线呈"S"形,接种1 d后有少量细胞贴壁,第3～6天为对数增殖期,第7天进入平台期。BMSCs经成骨诱导3周后茜素红染色细胞外基质中可见深红色团块状矿化结节;经成脂肪诱导4周后胞浆内充满"串珠状"红色脂滴。流式细胞仪检测BMSCs表面抗原CD29、CD44、CD90阳性,CD34和CD45阴性。结论应用7d龄SD乳鼠的改良全骨髓自然贴壁筛选法可以高效分离纯化出BMSCs。更多还原

【Abstract】 Objective To establish a simple and efficient method of separating and purifying BMSCs from neonatal SD rats through an improved system. Methods Six 7-day SD rats were sacrificed and bilateral femurs were dissected. The femurs were immersed in complete culture medium,and cut into pieces. The exposed bone marrow cavities were repeatedly syringed. The femur pieces and syringed bone marrow tissue suspension were transferred and shaken at high speed on a shaker for 3 minutes. The supernatant was inoculated in cell culture flasks and incubated at 37 ℃,5% CO₂. After 24 hours,half of the culture medium was changed to remove the non-adherent cells. After 48 hours, the whole medium was changed. Then the medium was changed every other day. The proliferation of the neonatal SD rat BMSCs was monitored using CCK-8 assay and growth curves were plotted accordingly. To examine the cell differentiation, alizarin red staining was performed three weeks after the osteogenic induction, and oil red "O" staining was performed four weeks after adipogenic induction was conducted. The expressions of BMSCs surface markers were identified by flow cytometry.Results The growth curve of BMSCs cells in the primary culture showed an "S" shape. After 24 hours of incubation,small amount of cells adhered to the bottle wall. Suspending blood cells were mostly cleared after changing the medium after 48 hours. The growth period was from the 3^rd to the 6^th day. After three days,the cells proliferated rapidly. After five days,colony fusion was about 90%. After the 1^st passage,the growth and proliferation of the cells were both rapid. The growth incubation period was short and cell adhesion was basically completed within 12 hours. Subsequently, the cells fully covered the flask bottom in about 2-3 days.At about the 7^th day,cells grew slowly and turned into a plateau. After 21 days of osteogenic induction,there were dark red mineralization nodules found in the extracellular matrix after Alizarin red staining. After 28 days of adipogenic induction,the cytoplasms were found filled with " bead-like" red lipid droplets. The results of flow cytometry showed that cell surface antigens CD29, CD44, CD90 were positive, while CD34 and CD45 were negative. Conclusion In this study,we separated and cultured BMSCs from 7-day neonatal rats using a modified whole bone marrow natural adherence screening method. By this method,a large number of BMSCs were obtained and purified,with a rapid proliferation rate.更多还原