兔外周血内皮祖细胞的分离培养、鉴定和体外标记

peripheral blood endothelial progenitor cells of rabbits

目的 研究新西兰大白兔外周血内皮祖细胞的分离、培养方法,对其进行功能鉴定,同时用增强型绿色荧光蛋白对细胞进行标记示踪,为后续实验研究做好准备.方法 ①以健康雄性新西兰大白兔为研究对象,密度梯度法分离兔外周血单个核细胞,采用专用的EGM-2 MV完全培养基对单个核细胞进行诱导分化培养,将其接种在人纤维连接蛋白包被培养板,动态观察细胞生长过程.②通过DiL标记的乙酰化低密度脂蛋白和FITC标记的凝集素UEA-1双染法鉴定血管内皮祖细胞,显示红色荧光的为吞噬了乙酰化低密度脂蛋白的细胞,绿色荧光为结合UEA-1的细胞,双染色为橙色荧光.③用携带绿色荧光蛋白基因的腺病毒液对培养7 d细胞进行感染,用荧光显微镜观察细胞绿色荧光蛋白表达情况.结果 细胞形态观察：新分离的骨髓单个核细胞呈圆形,第3～4天可观察到贴壁梭型细胞,第5～8天出现多个细胞团.乙酰化低密度脂蛋白和凝集素UEA-1双染法鉴定血管内皮祖细胞结果：在血管内皮祖细胞的胞质中,出现与乙酰化低密度脂蛋白结合的红色荧光聚集,阳性率达95%以上,与凝集素UEA-1结合率几乎达100%,2者双染色率达90%以上;腺病毒感染细胞后24 h即可表达EGFP,5 d表达最强,1～2周表达稳定,此后逐渐减弱;腺病毒感染MSCs效率约为95%.结论 从兔外周血中能成功地分离培养出具有血管内皮祖细胞特征的细胞群体;经Ad-EGFP感染后的EPCs可有效表达EGFP,且转染效率较高,是一种较好的EPCs标记方法.

Objective To investigate the isolation and culture of endothelial progenitor cells（EPCs） from rabbit peripheral blood and the identification of the cells.To explore the feasibility of using enhanced green fluorescent protein （EGFP） to label the cells in order to get ready for subsequent experiments.Methods Male healthy New Zealand white rabbits were selected.Total rabbit peripheral blood mononuclear cells （MNCs） were isolated by Histopaque density-gradient centrifugation and were suspended in endothelial basal medium supplemented with EGM-2 MV BulletKit and then the cells were plated on fibronectin-coated culture dishes.Process of cell growth was observed.EPCs were identified by Dil labeled acetylated low density lipoprotein and FITC labeled UEA-1 lectin.The cells showed red fluorescence were cells phagocytized acetylated low density lipoprotein,while those with green gluorescence were cells bind with BS-1,and the cells double stained showed orange fluorescence.The EPCs were infected by the adenovirus supernatant carrying EGFP,the expression of EGFP were detected by fluorescence microscope.Results Observation of cell morphous showed that new isolated mononuclear cells were round.Attached spindle-like cells were observed at 3 to 4 days＇ culture ,while cell clusters were observed at 5 to 8 days.Result of EPCs identification by Dil labeled acetylated low density lipoprotein and FITC labeled BS-1 lectin showed that in kytoplasm of EPCs,red fluorescent concentration bind with acetylated low density lipoprotein appeared,with the positive rate of over 95%.Combined rate with UEA-1 lectin nearly reached 100%.Double staining rate reached over 90%.The expression of EGFP began in 24 hours,reached maximum in 4-5 days,maintained stable from week one to week two after infection and then decreased.Transfection efficiency was about 95%.Conclusion Cell colony with the feature of EPCs can be isolated and cultured successfully from peripheral blood of rabbits.The EPCs that transfected by Ad-EGFP can express EGFP efficiently and the transfection efficiency is ralatively high,which proved this method is effective to label EPCs.