大鼠骨髓间充质干细胞的分离培养和生物学特性研究

目的 探讨体外分离培养大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的方法,并研究其生物学特性.方法 取大鼠股骨和胫骨,以Ficoll密度梯度离心法结合贴壁法分离纯化大鼠BMSCs,传代扩增,倒置显微镜进行细胞形态学观察,MTT法测定细胞的生长曲线,流式细胞仪细胞表面抗原.结果 原代分离的BMSCs,培养48 h开始贴壁,胞体由圆形变为椭圆形、多角形或短梭型;培养第12天,见胞体渐变为长梭型,并达90%单层融合;经传代扩增,细胞进一步纯化.7代以前,细胞在2 d内处于潜伏期,第3天进入对数生长期,第7天进入平台期;10代后增殖速度变慢;流式细胞仪鉴定BMSCs表而抗原,CD44、CD90、CD34阳性率分别为99.62%、95.13%、2.06%.结论 Ficoll密度梯度离心法结合贴壁法有效分离纯化大鼠BMSCs,且细胞生长稳定,增值能力活跃,具有MSCs的一般生物学特性.

Iprovement of culture method for bone marrow mesenchymal stem cells in vitro and study of biological characteristics of cultured cells

Objective To establish a simple and efiective method of isolation,puriftcation and culture of rat bone marrow mesenchymal stem cells(BMSCs)in vitro,and explore their biological characteristics. Methods BMSCs were isolated and purified by modified ficoll density gradient centrifugation combined with adherence culture method. The shape and growth curve of BMSCs were observed,and membrane antigens were detected with flow cytometric analyses. Results The primary cultured BMSCs adhered to plastic surface within 48 hours and exhibited oval,asteroid and fusiform shape. After cultured in DMEM-F12 medium containing 10%PBS for 12 days,the cells reached 90%confluence in single layer,and high purity with the uniform fibroblast like morphology.BMSCs were entered into latency after 2 days,convened into growing period after 3 days and entered into stationary phases after 7days.The results of FMS analysis indicated the positive rate of CD44 was 99.62%and of CD 90 was 95.13%and of CD34 was 2.06%.Conclusion Culture speed of BMSCs by modified ficoll density gradient centrifugation combined with adherence culture method can be used to isolate and purify BMSCs. Cultured BMSCs show stable and rapid growth in vitro and have general biological characteristics,which further make them as ideal seed cells for tissue engineering.