摘要
目的探讨荧光染料羧基荧光素二醋酸盐琥珀酰亚胺酯（CFSE）标记兔骨髓间充质干细胞（Rb—MSCs）的条件及其对Rb-MSCs生物学特性的影响。方法用全骨髓贴壁法分离纯化Rb—MSCs并进行鉴定；CFSE在不同条件下标记Rb—MSCs，流式细胞仪检测标记率及荧光强度；CCK．8法检测标记细胞的增殖能力，成骨成脂诱导检测标记细胞的分化能力，ELISA法检测标记细胞分泌血管内皮生长因子（VEGF）的能力。结果原代Rb-MSCs呈梭形，集落样生长；传代后细胞变为形态均一，排列有序的成纤维细胞样，流式细胞仪检测其表面标志物符合MSCs表型。与其它标记条件相比，10μmol／L终浓度CFSE标记Rb-MSCs10min，标记率达100％，且荧光强度高。标记细胞的增殖能力及分泌VEGF的能力与未标记细胞相比，差异无统计学意义（P〉0．05）；且标记细胞具有成骨及成脂的分化能力。结论CFSE标记Rb—MSCs是一种简单且高效的方法，适用于短期标记。且标记后Rb—MSCs的增殖能力、分化能力及分泌能力不受影响。
Objective To explore the experimental conditions of labeling rabbit bone marrow mes enchymal stem cells （RbMSCs） with 5,6carboxyfluorescein diacetic succinimidy] ester （CFSE） and to in vestigate the impact on the biological characteristics of RbMSCs in vitro. Methods RbMSCs were separa ted and purified by whole hone marrow adherent culture and then were identified by morphology and surface markers. RbMSCs were labeled with CFSE and the labeling effect was measured by flow cytometer. The proliferation capacity of labeled cells was detected with CCK8. The differentiation capacity of labeled cells was investigated by being induced to osteoblasts and lipoblasts. The capacity of labeled cells to secret vascu lar endothelial growth factor （VEGF） was detected by VEGF ELISA kits. Results The primary RbMSCs adhered in 48 h , being fusiform and colonylike growth. Subculture cells became fibroblastlike cells in or der with uniform configuration. Most （ above 98% ） cultured cells expressed the surface markers CD29 and CD44 except for CD45. Compared with other labeling conditions, 10 μmol/L final concentration of CFSE and 10 min was the best one with a 100% labeling rate and high fluorescence intensity. Compared with un labeled ceils , the ability of the labeled cells to proliferate and to secrete VEGF was not significantly de creased （ P 〉0.05）. Moreover, the labeled cells had osteogenic and adipogenie differentiation capacity. Conclusions It was a simple and efficient method to label RbMSCs with CFSE, especially in a shorttenn. The capacity of cell proliferation, differentiation, and secretion were not affected.
出处
《中国医师杂志》
CAS
2012年第11期1441-1444,共4页
Journal of Chinese Physician
基金
国家自然科学基金资助项目（30971235）
关键词
骨髓细胞
细胞学
间质干细胞
细胞学
细胞
培养的
染色与标砂方法
兔
Bone marrow cells/cytology
Mesenchymal stem cells/cytology
Cells, cultured
Stai-ning and labeling/methods
Rabbits
