BrdU标记人脐带间充质干细胞及对细胞生物学行为影响的体外研究

Effect of 5-bromo-2-deoxyuridine labeled umbilical cord mesenchymal stem cells on cell biology behaviors in vitro

目的:探讨5-溴脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine,Brd U)标记人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,h UC-MSCs)的方法及其对细胞存活、增殖以及凋亡的影响。方法:将h UC-MSCs分离培养并进行细胞表面标志物及多向分化潜能鉴定,取第5代细胞以终浓度分别为10、15、20、25μmol/L Brd U进行标记,采用空白Brd U作为对照组,分别培养24、48、72 h及96 h后,台盼蓝排斥实验检测细胞活力;MTT法检测细胞增殖能力;Annexin V-FITC检测细胞存活率及凋亡率,免疫组化法检测各组标记率。结果:台盼蓝排斥实验检测细胞活力都在96%以上,实验组与对照组比较均无统计学意义(不同浓度组间比较P=0.89,各时间点比较P=0.61,时间与不同浓度的交互作用P=0.89)。MTT结果显示Brd U对细胞增殖无抑制(P>0.05)。不同浓度标记组间细胞增殖率及凋亡率的差异(增殖率F=12.02,P<0.001,凋亡率F=6.14,P=0.009)。结论:Brd U标记h UC-MSCs是安全可靠的,Brd U标记h UC-MSCs的最佳剂量和时间为20μmol/L、72 h,适于h UC-MSCs在临床前实验研究的体内示踪。

Objective：To explore the method of using 5-bromo-2-deoxyuridine（BrdU） to label human umbilical cord mesenchymal stem cells（hUC-MSCs）,and to explore its effect on cell proliferation and apoptosis. Methods ：HUC-MSCs were isolated and cultured by direct adherent method, then the surface marker and potential ability of multi-direction differentiation were analyzed .The fifth passage of cells were labeled with BrdU at concentrations of 10, 15,20,25 μmol/L, excluding BrdU as control group. Cells were cultivated respectively for 24,48,72,96 h. Trypan blue testing was used to detect cell vitality, MTT method was used to analyze cell proliferation and AnnexinV-FITC assay was used to detect cell survival rate and apoptosis rate. Results ：Trypan blue testing showed that the living cells was more than 96% and there was no statistical significance between experimental group and control group（comparison among different concentration groups, P=0.89;comparison among different time points,P=0.61 ;interaction between time and different concentrations,P=0.89）. MTT method detected that there was no significant difference in proliferation activity between labeled cells and unlabeled cells （P〉0.05）. There were differences between cell proliferation and apoptosis rate （proliferation rate,F=12.02,P〈0.001 ;apoptosis rate,F=6.148,P=0.009）. Conclusion ：The tracing method of BrdU labeling hUC-MSCs is safe and effective. The best labeling concentration and time is 20 μmol/L and 72 h.