大鼠骨髓间充质干细胞体外CFSE染色标记的实验研究

CFSE fluorescence staining of rat bone marrow mesenchymal stem cells in vitro

关键词:  骨髓间充质干细胞  CFSE  荧光染料  细胞标记

Key words:  [Key words] bone marrow mesenchymal stem cells  CFSE  carboxyfluoresceindiacetate  cell labeling

摘要:

[摘要] 　目的　建立大鼠骨髓间充质干细胞（BMSCs）体外分离、培养、羟基荧光素二醋酸盐琥珀酰亚胺脂（CFSE）荧光标记的实验方法。方法　差速贴壁离心法分离纯化大鼠BMSCs，并进行表面标志物流式检测鉴定和成脂、成骨分化鉴定。CFSE标记BMSCs，荧光显微镜下观察BMSCs形态和标记率，台盼蓝染色检测CFSE对BMSCs存活率的影响，以筛选CFSE标记BMSCs的最佳条件。结果　第5代BMSCs的特异性表面标志物： CD29阳性，阳性率为90.10 %；CD90阳性，阳性率为96.61 %； CD45阴性，仅0.40 %表达。且BMSCs具有成脂和成骨分化能力。10 μmol/L CFSE作用15 min时，BMSCs的CFSE标记率几乎达到100 %，且对BMSCs的活性没有影响（P>0.05）。结论　利用差速贴壁法可获得纯度高、未分化性能强的BMSCs，BMSCs标记CFSE的最佳条件为10 μmol/L CFSE作用15 min。

Abstract:

[Abstract] Objective To investigate the method of isolation, cultivation and CFSE label of rat bone marrow mesenchymal stem cells (BMSCs). Methods BMSCs were isolated and expansion by adherent method. The specific surface markers of fifth generation BMSCs : CD29, positive rate was 90.10%. CD90, positive rate was 96.61%. CD45, negative rate was 0.40%, via flow cytometry, and differentiation. BMSCs labeled with CFSE were observed under fluorescence microscope and the labeling efficiencies were assessed. The effect of CFSE on the survival of BMSCS were tested by trypanblue. Results FCM detected that CD29 and CD90 were positively expressed, but CD34 was negatively expressed in BMSCs. Following induction, oil red O staining and alizarin red staining produced a strong reaction in cells. Nearly 100 % BMSCs were CFSE-labeled when BMSCs reacted with 10 μmol/L CFSE for 15 min. At the same time, the highest viability of BMSCs were observed(P>0.05). Conclusion BMSCs were obtained by adherent method. And staining with CFSE at 10 μmol/L for 15 min was the optimal condition for rat BMSCs labeling in vitro.