EdU体外标记人脂肪干细胞实验探究.doc

EdU体外标记人脂肪干细胞实验探究 ［摘要］目的:体外培养人脂肪干细胞(adipose-derived stem cells, ADSCs)并行EdU标记，并通过测试标记率及 标记后对细胞增殖分化的影响确定优化的标记时间及浓度 组合。方法：使用分别采5uM, 10uM, 20 nM, 50 uM的浓 度及12h, 24h进行标记标记ADSC,并以流式细胞仪精确测 定标记率。挑选出各12h及24h时优化标记浓度，并进行标 记后测定细胞活性，增殖及诱导分化实验。结果：P3代细胞 约90%以上表达表面标记CD90+, CD105+, CD44+。基本不表 达细胞表面标记CD45+, CD35+O通过各试验，得出最适浓度 组合10uM, 12h,对进一步研究脂肪干细胞在辅助脂肪移植 中的作用具有指导意义。结论：EdU是标记方法简单有效， 体外最佳标记浓度组合是10 pM, 12ho ［关键词］脂肪干细胞；细胞治疗；EdU；干细胞标记； 细胞不踪 ［中图分类号］R622 Q813. 1 ［文献标识码］A ［文章编 号］1008-6455 (2013) 01-0043-05 Experimental study on the effect of labeling adipose derived stem cells with EdU ZHANG Yi, WANG Ke-ming, LI Fa-cheng, WANG Shu-jie, LI Tai-ying, LIUXin-hai, LIU Mei-chen, ZHAO Xiao-hui, MA Ji-guang (Special Medical Department , Plastic Surgery Hospital , Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100144, China) Abstract： Objectives To est ablish the best met hod and optimal concentrantion and optimal time for labeling ADSC with thymidine analog , 5-ethynyl-2-deoxyuridine ( EdU )? Methods ADSC a/t passage 2 ? 4 were labeled with EdU by diverse concentration (5 u M, 10 u M, 20 u M, 50 u M) and diverse time (12h, 24h) . After labeled, the cell were stained by ALEXA-555 analyzed by fluorescence microscopy and flow cytometer. The impact of EdU on the growth of ADSCs was examined by trypan blue exclusion, methylthiazoly tetrazoli u M ( MTT ) assay and assess multipie differentiation potential of ADSC using in vitro osteogenic and adipogenic induetion. Results Labeling with EdU in vitro can be easily performed, And labeling stem cells with the concentration of 10 P M with 12h is the optimal concentration and time. The optimal concentration and time of labeling has little impact on the grow th of ADSC or on the differe ntiation and can therefore be used for ADSC trading? Conclusion The lebeling method with EdU is simple but efficacious, and the optimal group is labeling with the concentration of 10 y M and the time with 12h. Key words : adipose-derived stem c