兔骨髓间充质干细胞的分离、培养、鉴定及Dil荧光标记

摘要 目的　探讨兔骨髓间充质干细胞(MSCs)体外分离培养、表型鉴定和标记的方法。方法　采用密度梯度离心法及贴壁分离筛选法分离培养MSCs；采用免疫细胞化学方法检测细胞表面标志抗原CD29，CD106的表达，进行表型鉴定；DiI标记第3代MSCs,观察标记效率。结果　体外培养的原代MSCs48h内可见少量贴壁细胞，7-8d达到90%汇合；免疫细胞化学方法检测细胞表面标志抗原CD29，CD106为阳性；DiI进行细胞标记后,荧光显微镜下见所有MSCs均被标记为红色荧光,提示DiI标记法敏感性好,标记效率高。结论　此培养方法能在短时间内获得大量MSCs,操作简单,成功率高,可以作为培养兔MSCs的常规方法，为构建组织工程尿道提供充足的种子细胞，并进一步用于治疗重度尿道下裂、尿道下裂残废和较长的后尿道狭窄等顽症。

Culture,identification, and labeling of rabbit mesenchymal stem cells in vitro

Abstract Objective To establish a method of isolation, culture, identification and labeling rabbit mesenchymal stem cells (MSCs) in vitro. Methods MSCs were isolated and cultivated by density gradient and adherent methods. The expressions of CD29 and CD106 of cells were analyzed by using Immunocytochemistry. The third generation of MSCs were labeled by DiI，the labeling efficiency was detected. Results　Primary cultured MSCs adhered to plastic surface within 48h and reached 90% confluence within 7-8 d . MSCs expressed CD29 and CD106. All of the MSCs after labeling by DiI showed red fluorescence by immunofluoroscope. DiI labeling was sensitive and highly efficient to MSCs. Conclusion The method is simple and easy to isolate and cultivate MSCs and it can serve as a routine method. The culturing cells could be used in urethral construction of tissue engineering and then could be used to treat severe hypospadias、hypospadias cripples and urethral stricture in the future.