摘要
目的探讨体外分离、培养及荧光染料CFSE标记大鼠间充质干细胞(MSCs)的方法。方法Wistar大鼠10只。用密度梯度离心及贴壁法相结合的方法分离纯化大鼠MSCs并进行鉴定。荧光染料CFSE标记MSCs,荧光显微镜检测标记率,CCK-8法检测标记细胞的增殖能力。结果原代MSCs72h贴壁,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。CFSE标记当天,荧光显微镜下大鼠MSCs呈明亮的绿色荧光,标记率达100%,随着培养时间延长,荧光强度逐渐减弱,标记率随之下降,标记4周后,标记率下降至78%;标记细胞的增殖能力与未标记细胞相比,差异无统计学意义(P>0.05)。结论利用密度梯度离心法结合贴壁法可以获得比较纯的MSCs;CFSE短期标记MSCs效率很高,且标记细胞的增殖能力不受影响,但荧光强度及标记率随着时间延长而下降。
Objective To investigate the method of isolation, purification and culturing of rat bone marrow mesenchymal stem cells （MSCs） and to explore the feasibility of labeling MSCs with 5,6-carboxyfluorescein diacetic succinimidyl ester （CFSE）in vitro. Methods obtained from Wistar rats＇ bone marrow were isolated and purified in vitro by Percoll density gradient centrifugation combined with adherent method, then cultured and propagated. The cultured cells were identified by surface markers （CD29, CD34, CD44 and CD45） via flow cytometry （FCM）. The MSCs labeled with CFSE were observed under microscope and the labeling efficiency was assessed by fluorescence microscope. The proliferation ability of these cells was tested by CCK-8 and compared with that of non-labeled MSCs. Results The primarily cultured MSCs were spindle in shape, and were adherent after 72 hours, and then grew into small clones. After subculturing, MSCs gained uniform shape and similar growth pattern. 90% of cultured cells express surface markers CD29 and CD44, but CD34 and CD45. The initial labeling efficiency was as high as 100%. The labeling efficiency and fluorescence intensity decreased with elapse of time. There was no statistical difference in proliferation ability between the labeled and non-labeled MSCs （P〉0.05）. Conclusions Highly purified MSCs can be obtained by Percoll density gradient centrifugation combined with adherent method. CFSE can obtain high labeling efficiency for a short time, and the labeled MSCs maintained the same proliferation ability as non-labeled MSCs. However, along with passage of time, the fluorescence intensity and labeling efficiency decreased.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2007年第5期464-466,共3页
Medical Journal of Chinese People's Liberation Army
基金
广东省科技计划项目(2006B35901003)
关键词
干细胞
二醋酸盐琥珀酰亚胺酯
生物学标记
细胞增殖
stem calls
5,6-carboxyfluorescein diacetate succinimidyl ester
biological markers
cell proliferation
