SD大鼠骨髓间充质干细胞体外培养、表型鉴定及标记

目的：探讨SD大鼠骨髓间充质干细胞(MSCs)体外分离培养、表型鉴定和标记的方法，为进一步研究MSCs干预器官移植的免疫排斥奠定基础。方法：采用直接贴壁法分离培养MSCs；通过流式细胞术检测细胞表面标志抗原CD90、CD45的表达率对培养的 MSCs进行表型鉴定；DAPI标记第3代MSCs，观察标记效率。结果:体外培养的原代MSCs 48 h内可见有少量贴壁细胞，7～10 d达到90%汇合；流式细胞术检测，第3代MSCs表面标记物CD90、CD45的阳性率分别为99.8%、6.8%, 提示MSCs表达CD90而不表达CD45；用DAPI进行细胞标记后，荧光显微镜下见所有MSCs均已被标记蓝色荧光，提示DAPI标记法敏感性好，标记效率高。结论：贴壁培养法是一种简便易行的培养MSCs方法，操作简单，成功率高，可以作为培养SD大鼠MSCs的常规方法；DAPI标记可作为标记MSCs的一种有效手段。

Culture, identification of phenotype, and labeling of mesenchymal stem cells in vitro in SD rats

Objective To investigate the methods of isolation,culture,identification and labeling of mesenchymal stem cells(MSCs) in vitro and lay a foundation for further study on intervention of MSCs on immunologic rejection of organ transplantation. Methods MSCs were isolated and cultivated by adherent methods . The expressions of CD90 and CD45 of cells were analyzed by using flow cytometry in order to identify MSCs.The third generation of MSCs were labeled by DAPI,the labeling efficiency was detected.Results Primary cultured MSCs adhered to plastic surface within 48 h and reached 90% confluence within 7-10 d .Flow cytometry showed that the positive rates of CD90 and CD45 of MSCs at third generation were 99.8% and 6.8%. MSCs expressed CD90 but no CD45.All of the MSCs after labeling by DAPI showed blue fluorescence by immunofluoroscope. DAPI labeling was sensitive and highly efficient to MSCs.Conclusion Adherent method is simple and easy to isolate and cultivate MSCs and it can serve as a routine method.DAPI labeling can be used as a efficient method to label MSCs.