5-乙炔基-2’脱氧尿嘧啶核苷标记大鼠骨髓间充质干细胞的有效性

Efficacy of labeling rat bone marrow mesenchymal stem cells with 5-ethynyl-2’-deoxyuridine

背景:深入准确地研究骨髓间充质干细胞在体内外增殖、分化的规律,寻找安全、有效、灵敏度高、特异性强、简便的标记活细胞的方法至关重要。目的:探索5-乙炔基-2’脱氧尿嘧啶核苷标记骨髓间充质干细胞的有效性及对其增殖能力的影响。方法:全骨髓贴壁法培养扩增SD大鼠骨髓间充质干细胞至第3代。将细胞以5×107L-1浓度接种于96孔板中,掺入法加入终浓度为0,10,20,50μmol/L5-乙炔基-2’脱氧尿嘧啶核苷,分别在培养24,48和72h检测5-乙炔基-2’脱氧尿嘧啶核苷标记细胞的阳性率、标记后的细胞形态,并绘制标记细胞的生长曲线。结果与结论:各浓度组标记率均随标记时间延长而升高(P<0.05);各时间点标记率随标记浓度的升高而增加,但其差别无显著性意义(P>0.05)。10μmol/L5-乙炔基-2’脱氧尿嘧啶标记骨髓间充质干细胞72h即可获得良好的标记率。标记前后各组间充质干细胞生长曲线均呈"S"形。说明在实验浓度范围内应用5-乙炔基-2’脱氧尿嘧啶核苷体外标记骨髓间充质干细胞有效,不影响细胞的增殖,且10μmol/L5-乙炔基-2’脱氧尿嘧啶核苷标记骨髓间充质干细胞72h即可获得良好的标记率。

BACKGROUND： A safe, efficient, and convenient labeling method with high sensitivity and strong specificity is needed to understand the proliferation and differentiation of bone marrow mesenchymal stem cells （BMSCs） in vitro and in vivo. OBJECTIVE： To investigate the efficacy of labeling BMSCs with 5-ethynyl-2’-deoxyuridin （EdU） and its effects on cell proliferation. METHODS： BMSCs were isolated by adherence method from bone marrow of rats and cultured to the third generation. BMSCs were seeded into 96-well plate with concentration of 5×107/L. BMSCs were labeled with EdU at different concentrations （0, 10, 20 and 50 μmol/L）. The EdU-labeling positive rates and morphology of BMSCs in each group were detected at 24, 48 and 72 hours of culture, and the cell growth curves were obtained. RESULTS AND CONCLUSION： The EdU-labeling positive rates were gadually increased in each group as time went on （P 0.05）, and the differences in the increased EdU-labeling positive rates were not significant （P 0.05）. Good EdU-labeling positive rates could be obtained in the 10 μmol/L group at 72 hours of culture. The BMSCs growth curves of each group before and after labeling were ＂S＂-shaped. This suggested that application of EdU labeling within the experimental concentrations for BMSCs in vitro is efficient and safe without effects on cell proliferation. Good labeling rate of BMSCs can be obtained through labeling with EdU in 10 μmol/L for 72 hours.