三种示踪技术标记人脂肪组织来源干细胞的对照研究

摘要

目的 比较3种不同的荧光示踪技术标记人脂肪组织来源干细胞(ASCs)的效率,以寻找脂肪组织来源干细胞最佳的标记示踪方法.方法 吸脂术获取人脂肪组织,胶原酶消化法后体外贴壁法培养,获得的长梭形细胞经鉴定为脂肪组织来源干细胞.分别使用5μlDiI,10μg/ml的脱氧尿苷BrdU及50 MOI的携带绿色荧光蛋白(GFP)的重组腺病毒进行标记,荧光显微镜观察不同时间点及不同代数的脂肪干细胞的标记效率及形态变化.结果 成功分离获得ASCs,经鉴定其表达间充质干细胞表面标志,并能被成功实现成脂、成骨及成软骨的诱导分化.DiI标记ASCs 48 h后,荧光显微镜下观察可见100%细胞浆呈现红色荧光,胞核未染,保持了良好的正常形态.但细胞传代后荧光衰减迅速.10μg/ml BrdU标记ASCs 48h后,90%的胞核呈绿色荧光,传代后胞核荧光逐渐衰减.携带GFP重组腺病毒转染ASCs 24 h后胞浆内即可见绿色荧光,5 d后90%以上的细胞呈现绿色荧光.反复传代后未见明显的荧光衰减.结论 DiI,BrdU及携带GFP的重组腺病毒均能有效的标记人脂肪组织来源干细胞.DiI示踪技术为细胞膜标记,BrdU为细胞核标记,两项技术均操作简单,但传代后衰减迅速,适合于短期标记示踪.携带GFP的腺病毒标记方法较为复杂,但反复传代后仍不衰减,适合于长期的标记示踪观察.%Objective To explore the optimal methods for labeling human adipose-derived stem cells (ASCs). Methods ASCs were isolated by collagenase digestion and density gradient centrifugation, and their cell surface markers and ability to differentiate into the adipogenic, chondrogenic, and osteogenic lineages were examined in vitro. Three different cell labeling methods, namely 5μ Dil, 10 μg/ml BrdU and 50 MOI adenovirus carrying GFP, were used for ASC labeling, and the labeling efficiency were compared at different time points and in different passages using fluorescent microscope. Results The isolated ASCs were capable of differentiating into adipogenic, osteogenic, chondrogenic lineages with positive stem cell marker expression. At 48 h after Dil staining, 100% of the ASCs emitted red fluorescence in the cytoplasm with fluorescent-negative nuclei, but the fluorescence intensity declined quickly after cell passaging. With 10 μg/ml BrdU, 90% of the cells showed green fluorescence in the cell nuclei at 48 h after the labeling, but the positivity rate also decreased gradually after cell passaging. Cell labeling with GFP adenovirus showed more stable labeling efficiency, and green fluorescence was detected at 24 h after labeling, and even till 5 days later more than 90% of the ASCs remained positive without an obvious attenuation of the fluorescent intensity even after cell passaging. Conclusions All the 3 techniques are applicable for labeling ASCs. Cell labeling with Dil and BrdU can be convenient and economic and well serve the purpose of short-term labeling. Adenovirous carrying GFP gene is the optimal choice for long-term ASC tracing.