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，24 h 后开始贴壁，2 d 后开始长出伪足，7～8 d 细胞融合达90％，纯化后 BMSCs 呈梭形，24 h 细胞贴壁率可达99％，前4代细胞传代周期为7 d，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 methods of separate,culture and identification of rat bone marrow mesenchymal stem cells.Methods By adherent separation method,bone marrow mesenchymal stem cells were isolated and purified from 3-4 weeks old SD rat,then cell growth was observed,respectively by im-munofluorescence staining,flow cytometry and DAPI nuclear staining method for identification of BMSCs purity and activity.Results Primary BMSCs was round,uniform in size and light in transmission.After 24h begin to adherent,2 d began to grow following pseudopodia,90% of the cells were fused on 7-8 d, and purified BMSCs werefusiform.24 h cell adherent rate was 99% and before the 4th generation cell cycle was 7 days.After subculture,cycle gradually shortened.The results of surface markers of the third gener-ation MSCs showed thatthe positive rate of CD29 was 98.9%,and the positive rate of CD44 was 73.3%. DAPI labeled BMSCs,the nucleus can see bright blue fluorescence,labeling rate of 100%.Conclusion Adherent separation of rat BMSCs have high purity and strong vitality of BMSCs,and the third generation of BMSCs shows stable biological character.
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