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目的探索大鼠骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)的分离、培养及鉴定方法。方法采用贴壁分离方法,分离纯化3~4周龄SD大鼠的骨髓间充质干细胞,观察细胞生长状况,分别用免疫荧光染色、流式细胞仪鉴定及4′,6-二脒基-2-苯基吲哚(DAPI)标记细胞核的方法鉴定BMSCs纯度及活性。结果原代BMSCs呈圆形、大小均一,透光性可,24h后开始贴壁,2d后开始长出伪足,7~8d细胞融合达90%,纯化后BMSCs呈梭形,24h细胞贴壁率可达99%,前4代细胞传代周期为7d,之后传代周期逐渐缩短。第3代BMSCs的表面标志物CD29阳性表达率为98.9%,CD44阳性表达率为73.3%。DAPI标记的BMSCs胞核可见明亮蓝色荧光,标记率达100%。结论贴壁分离法分离大鼠BMSCs,可得到纯度较高、活力强的BMSCs,第3代BMSCs生物学性状稳定。
Objective To explore the isolation, culture and identification of rat bone marrow mesenchymal stem cells (BMSCs). Methods Adherent separation was used to separate and purify bone marrow mesenchymal stem cells of 3- to 4-week-old Sprague-Dawley rats. The growth of the cells was observed by immunofluorescence staining, flow cytometry and 4 ’, 6-diamidino- 2-phenylindole (DAPI) labeled nuclei were used to identify the purity and activity of BMSCs. Results Primary BMSCs were round, uniform in size and translucent. After 24 hours, the adherent BMSCs began to attach. After 2 days, pseudopodia began to grow. The fusion of BMSCs was 90% after 7-8 days. The rate of up to 99%, the first 4 generations of cell passage cycle 7d, after which the passage cycle gradually shortened. The third generation BMSCs surface markers CD29 positive rate was 98.9%, CD44 positive rate was 73.3%. DAPI labeled BMSCs nuclei showed bright blue fluorescence, labeling rate of 100%. Conclusion BMSCs isolated by adherent separation can obtain higher purity and higher viability of BMSCs. The third generation BMSCs have stable biological characteristics.