大鼠骨髓内皮祖细胞SPIO标记及检测

摘要

目的:研究大鼠骨髓内皮祖细胞(endothelial progenitor cells,EPCS)的分离、培养、SPIO标记,SPIO对EPCS的标记效果及其对细胞存活、增殖以及凋亡的影响,为进一步的实验研究奠定基础.方法:SD大鼠体重120～150 g,采用梯度密度离心法和贴壁法获取大鼠EPCS,采用25μg/ml SPIO对其标记,比较标记和未标记的EPCS.普鲁士蓝染色观察铁标记率,台盼蓝检测细胞活力,MTT法检测细胞增殖力,Calcein-AM/PI染色检测细胞活性及细胞膜完整性,AO/PI染色检测细胞凋亡率.结果:分离培养EPCS 7～10 d,细胞集落相互连接,呈典型的铺路石样外观.普鲁士兰染色显示SPIO(25 μg/ml,24 h)对细胞的标记率接近94%;比较SPIO标记及未标记的EPCS,细胞活力分别为(94.34±0.25)%和(95.33±0.31)%;MTT法检测两组间相比差异无统计意学义(P>0.05);Calcein-AM/PI染色检测细胞的活性和膜完整性,存活率分别为96%和97%;AO/PI染色两组细胞的凋亡率均为5%.结论:采用梯度离心法从骨髓中分离单个核细胞,经诱导培养可实现内皮祖细胞的分离培养.25 μg/ml浓度的SPIO细胞标记率高,对细胞毒性小,且不影响EPCS的活力、增殖及凋亡.%Objective: To study the isolation, culture, SPIO mark of the rat bone marrow progenitor cells (endothelial progenitor cells, EPCS), and observe whether there is any effect on cellular viability, proliferation and apoptosis. which lay the foundation for future experimental studies. Methods: The EPCS were collected from hone marrow of rats (weighing120-150 g) and cultured. EPCS were obtained by density gradient centrifugation and adhesive-screening methods and labeled with 25 μg/ml SPIO. Marked and unlabeled groups were compared. Prussian blue staining was used to identify labeling index, Trypan blue staining was employed to detect cell vitality, MTT assay was utilized to detect proliferation activity of stem cells, Calcein-AM/PI staining cell was detected cell activity and membrane integrity, AO/PI staining was detected cell apoptosis. Results: Cell Colonies were connected, showing the typical cobblestone appearance on the 7-10 d.Prussian blue staining showed that SPIO(25 μg/ml, 24 h) of cells labeled rate of close to 94％; compared SPIO labeled and unlabeled EPCS. cell viability was 94.34％±0.25％ and 95.33％±0.31％; MTT assay detected that there was no statistical difference between the groups compared with Italy in righteousness (P＞0.05); Calcein-AM/PI staining cell activity and membrane integrity, survival rates were 96％ and 97％; AO/PI staining cells withered groups death rate was 5％. Conclusion:Cradient centrifugation from bone marrow mononuclear cells can be achieved by induction of endothelial progenitor cells ,isolated and cultured; 25 μg/ml high concentrations of SPIO cell labeling dose not affect the EPCS viability, proliferation and apoptosis.