两种分离培养小鼠骨髓来源内皮祖细胞方法的比较

目的 比较密度梯度离心法及全骨髓培养法分离培养内皮祖细胞的差异。方法 取4周雄性近交系C57BL/6J小鼠骨髓,分别使用密度梯度离心法及全骨髓培养法培养,观察细胞贴壁情况和细胞形态,并行DiI acLDL及FITC UEA I双染、vWF、eNOS及细胞表面标志检测。结果 密度梯度离心法培养细胞可形成典型铺路石样改变及形成血管样结构;而全骨髓培养法贴壁细胞形态多样,较多呈长梭形铺展生长,部分细胞呈类圆形及纺锤形。比较两种方法培养细胞摄取DiI acLDL、结合FITC UEA I双阳性率以及vWF、eNOS及细胞表面标志表达阳性率,差别均有统计学意义（P〈005）。应用密度梯度离心法,随着培养时间延长,表达CD34、CD133及FLk 1细胞逐渐增多（P〈005）。结论 密度梯度离心法及全骨髓培养法在EGM 2MV培养体系下均可培养出内皮祖细胞,但密度梯度离心法较全骨髓培养法培养的内皮祖细胞纯度高。

Comparison between Two Culture Mothods of Endothelial Progenitor Cells Derived from Mice Bone Marrow

Objective To compare the effects of whole bone marrow culture and density gradient centrifugation in isolation and culture of endothelial progenitor cells （EPCs） derived from mice bone marrow. Methods Bone marrow was obtained from healthy C57BL/6J mice and isolated and cultured by whole hone marrow culture and density gradi- ent centrifugation method. And then, the change of cell morphology was observed and the abilities of uptaking acLDL and binding UEA-I, the expression of vWF, eNOS and cell-surface markers were detected. Results Primary cell culture with density gradient centrifugation method showed the characteristics of forming capillary structure and ＂cobble- stone＂ morphology. And under whole bone marrow culture, multiple cell morphology was showed, including long fusiform, similar circular and spindle shapes. Cells cultured with density gradient centrifugation method had higher rate in acLDL and UEA-I double positive cells and expression of vWF and eNOS than those of whole bone marrow culture （P〈0. 05）. Also, there were differences between two mothods in the expression of cell surface markers （P〈0. 05）. With the method of density gradient centrifugation, the cells expressed CD34, CD133 and Flk-1 increased with culture time （P〈0.05）. Conclusion EPCs could be obtained from mice bone marrow using these two methods under the culture system of EGM-2MV, and the method of density gradient centrifugation is an optimal way to acquire murine mar- row-derived EPCs with high-purity.