摘要
目的探讨活体染料羧基荧光素乙酰乙酸(CFSE)标记大鼠骨髓间质干细胞(MSCs)的可行性和最适条件。方法清洁型SD大鼠20只,用密度梯度离心结合贴壁法分离、纯化、培养大鼠MSCs,取长满25cm×25cm培养瓶瓶底的第3代大鼠MSCs,用2.5,5,10,20,40μmol/L的CFSE分别作用1,5,10,15,20min后染色,通过观察染色后细胞荧光强度和细胞贴壁率筛选出CFSE标记大鼠MSCs的最佳标记时间和标记浓度。采用MTT法检测最佳条件CFSE标记的大鼠MSCs的增殖能力。结果浓度10μmol/L的CFSE在37℃下孵育10min为MSCs染色、观察和研究的最佳条件。此条件下,CFSE标记的MSCs的增殖能力不受影响。结论活体染料CFSE是一种标记率高、细胞毒性小、操作简便的标记大鼠MSCs的新方法,浓度10μmol/L的CFSE在37℃下孵育10min为CFSE标记大鼠MSCs的最佳条件。
Objective To investigate the feasibility of labeling mesenchymal stem cells（MSCs） of rats with 5,6,2-carboxyfluorescein diacetate suecinimidy ester（CFSE）and the optimal condition. Methods MSCs obtained from 20 SD rats＇ bone marrow were isolated, purified and cultured in vitro with Percoll density gradient centrifugation combined with adherent method. MSCs of the third generation MSCs filling in the floor of 25cm^2 cultured bottles were incubated with 2.5,5,10,20, and 40μmol/L of CFSE for 1, 5,10,15, and 20 min respectively. The optimal concentration and labeling time of CFSE were indentified by detecting the fluorescence intensity and adhesive rate of labeled MSCs. The proliferative activity of MSCs labeled by CFSE under the fittest condition was tested by MTT method. Results Incubation with 10μmol/L of CFSE under 37 ℃ for 10 minutes was the optimal condition for MSCs labeling. The labeled MSCs maintained the same proliferative ability as the non- labeled MSCs under the optimal condition. Conclusion With the advantages of high rate of dyeing, low cell toxicity, safety and convenience,CFSE can be used as a new method to label the rat＇s MSC. Incubation with 10μmol/L of CFSE under 37℃ for 10 minutes is the optimal condition for MSCs labeling.
出处
《福建医科大学学报》
2008年第6期482-486,共5页
Journal of Fujian Medical University
基金
教育部博士学科点科研基金(2006E+10)
关键词
荧光素
乙酰乙酸盐类
骨髓细胞
间质干细胞
染色与标记
大鼠
fluorescein
acetoacetates
bone marrow cells
mesenchymal stem cells
staining and labeling
rats, sprague-dawley
