CAJ | 학술논문

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,10 μmol/L终浓度CFSE标记Rb-MSCs 10 min,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.
목적 탐토형광염료최기형광소이작산염호박선아알지(CFSE)표기토골수간충질간세포(Rb-MSCs)적조건급기대Rb-MSCs생물학특성적영향.방법 용전골수첩벽법분리순화Rb-MSCs병진행감정；CFSE재불동조건하표기Rb-MSCs,류식세포의검측표기솔급형광강도；CCK-8법검측표기세포적증식능력,성골성지유도검측표기세포적분화능력,ELISA법검측표기세포분비혈관내피생장인자(VEGF)적능력.결과 원대Rb-MSCs정사형,집락양생장；전대후세포변위형태균일,배렬유서적성섬유세포양,류식세포의검측기표면표지물부합MSCs표형.여기타표기조건상비,10 μmol/L종농도CFSE표기Rb-MSCs 10 min,표기솔체100％,차형광강도고.표기세포적증식능력급분비VEGF적능력여미표기세포상비,차이무통계학의의(P＞0.05)；차표기세포구유성골급성지적분화능력.결론 CFSE표기Rb-MSCs시일충간단차고효적방법,괄용우단기표기.차표기후Rb-MSCs적증식능력、분화능력급분비능력불수영향.
Objective To explore the experimental conditions of labeling rabbit bone marrow mesenchymal stem cells (Rb-MSCs) with 5,6-carboxyfluorescein diacetic succinimidyl ester (CFSE) and to investigate the impact on the biological characteristics of Rb-MSCs in vitro.Methods Rb-MSCs were separated and purified by whole bone marrow adherent culture and then were identified by morphology and surface markers.Rb-MSCs were labeled with CFSE and the labeling effect was measured by flow cytometer.The proliferation capacity of labeled cells was detected with CCK-8.The differentiation capacity of labeled cells was investigated by being induced to osteoblasts and lipoblasts.The capacity of labeled cells to secret vascular endothelial growth factor (VEGF) was detected by VEGF ELISA kits.Results The primary Rb-MSCs adhered in 48 h,being fusiform and colony-like growth.Subculture cells became fibroblast-like cells in order with uniform configuration.Most (above 98％) cultured cells expressed the surface markers CD29 and CD44 except for CD45.Compared with other labeling conditions,10μmol/L final concentration of CFSE and 10 min was the best one with a 100％ labeling rate and high fluorescence intensity.Compared with unlabeled cells,the ability of the labeled cells to proliferate and to secrete VEGF was not significantly decreased (P ＞ 0.05).Moreover,the labeled cells had osteogenic and adipogenic differentiation capacity.Conclusions It was a simple and efficient method to label Rb-MSCs with CFSE,especially in a short-term.The capacity of cell proliferation,differentiation,and secretion were not affected.