DAPI标记的骨髓间充质干细胞体内外示踪实验研究

目的:研究随着时间延长,DAPI标记间充质干细胞(MSCs)的标记率变化。方法:分离培养纯化成年大鼠骨髓MSCs,DAPI标记,不同时点荧光显微镜观察,计算MSCs中DAPI阳性率。将标记的MSCs移植到缺血下肢模型动物骨骼肌中,不同时点取材,冰冻切片,观察移植细胞。结果:标记当天,荧光镜下MSCs胞核呈明亮蓝色荧光,标记率接近100%,随着培养时间延长,标记率逐渐下降。移植1周和2周组织切片中,可见密集移植MSCs,3周以后,可检出的DAPI阳性细胞明显减少。结论:DAPI短期标记细胞效率很高,但标记率随着时间延长而迅速下降。

Tracing study of DAPI Labeled Mesenchymal Stem Cells in Vivo and in Vitro

Objective To explore labeling efficiency of DAPI labeled mesenchymal stem cells(MSCs) in vivo and in vitro,and provide technical foundation for the study of cell fate determination in the course of MSCs transplantation repairing ischemic diseases.Methods DAPI was added to the culture medium at a final concentration of 50 μg/ml for 30 minutes,when the cultured MSCs in passage 3 reached 50% confluency.For in vitro study,MSCs were collected during the initial period and after1,2,3 and 4 weeks.The Intensity and pattern of fluorescence,as well as the labeling efficiency,were evaluated just after labeling and again after 1,2,3 and 4 weeks.The scale was calculated based on the fraction of positive MSCs to the total MSCs attached to the bottom.For in vivo study,labeled MSCs were transplanted into ischemic hind limb in rats,and samples were collected 1,2,3 and 4 weeks after transplantation.These tissue sections were observed under the same microscope with the same filter,and the numbers of labeled MSCs were evaluated.Results DAPI achieved outstanding initial labeling efficiency for the cultured MSCs(all 100%).However,the labeling efficiency after 3,4 weeks decreased to 20% and 5% for DAPI in vitro,respectively.Intensive labeled MSCs in tissue sections were identified 1,2 weeks after MSCs transplantation,the numbers of labeled MSCs after 3 weeks decreased obviously.Conclusion An advantage of DAPI is the high labeling efficiency via simple procedure.The labeled MSCs were easily detected,and the initial labeling efficiency was 100%.However,disadvantage of DAPI is the loss of labeling intensity due to cell proliferation and division.DAPI may be used only for quantitative analysis for a short period.