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

    目的:探讨5-溴脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine,BrdU)标记人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)的方法及其对细胞存活、增殖以及凋亡的影响。方法:将hUC-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、25 ?滋mol/L BrdU进行标记,采用空白BrdU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;AnnexinV-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BrdU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:BrdU标记hUC-MSCs是安全可靠的,BrdU标记hUC-MSCs的最佳剂量和时间为20 ?滋mol/L、72 h,适于hUC-MSCs在临床前实验研究的体内示踪。

    Abstract:

    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 ?滋mol/L,excluding BrdU as control group. Cells were culti-vated respectively for 24,48,72,96 h. Trypan blue testing was used to detect cell vitality,MTT method was used to analyze cell pro-liferation 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(compar-ison among different concentration groups,P=0.89;comparison among different time points,P=0.61;interaction between time and dif-ferent 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 ef-fective. The best labeling concentration and time is 20 μmol/L and 72 h.

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高 黎,祝 华,张 荣,王莉佳,邹 琳,符 州. 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[J].重庆医科大学学报,2014,38(11):1617-1621

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  • 在线发布日期: 2015-11-03
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