【摘要】 背景:培养出生长状态良好、数量足够多的大鼠骨髓间充质干细胞是其作为种子细胞在组织工程等领域广泛应用的重要前提。目的:寻求快捷有效的大鼠骨髓间充质干细胞体外培养条件,观察培养的间充质干细胞生物学特性。方法:全骨髓法体外分离培养大鼠骨髓间充质干细胞,贴壁筛选法进行纯化,倒置相差显微镜下观察细胞形态及生长特征,免疫荧光分析细胞骨架,MTT法绘制细胞生长曲线,流式细胞仪检测其表面标记。结果与结论:分离培养的细胞呈长梭形或多边形;细胞生长曲线呈S形;微丝免疫荧光染色结果显示,培养的骨髓间充质干细胞具有良好的细胞骨架系统。第3代骨髓间充质干细胞CD29、CD44、CD71、CD90均呈阳性表达,而CD13、CD34、CD45、CD133呈阴性。提示,经全骨髓贴壁法体外分离培养的细胞在形态学和细胞表面标志物表达方面具有干细胞生物学特性,经鉴定为骨髓间充质干细胞,其第3代活性最佳,可用于后续实验。更多还原

【Abstract】 BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are widely used as seed cells in tissue engineering and other fields. Therefore, well growth and enough number of rat BMSCs are important for above-mention studies. OBJECTIVE: To explore the suitable primary culture condition of rat BMSCs in vitro, and to observe the growth property of BMSCs. METHODS: BMSCs were separated using whole bone marrow culture method and purified using attachment method. Morphology and growth characteristics were observed under inverted phase contrast microscope and cytoskeleton was detected by immunofluorescence. Growth curve was drawn using MTT method. Expression of BMSC surface antigens was detected by flow cytometer. RESULTS AND CONCLUSION: After isolation and culture, cells developed a spindle or polygonal appearance. Growth curve was S-shaped, and the cultured BMSCs possessed normal morphology and cytoskeleton. At the third passage, BMSCs were positive for CD29, CD44, CD71, CD90, but negative for CD13, CD34, CD45, CD133. Cells isolated and cultured by the adherence separation method in this study are identified as rat BMSCs by morphology and surface antigens, which have the biological characteristics of BMSCs.更多还原