小儿骨髓内皮细胞和内皮祖细胞的培养及鉴定

Isolation and Culture of Human Endothelial Cells and Their Progenitor Cells from Children's Bone Marrow

目的探索小儿骨髓内皮细胞和内皮祖细胞的分离培养,为组织工程血管、补片或带瓣管道的制备找新的种子细胞来源。方法采集小儿骨髓,梯度密度离心法分离单核细胞,内皮细胞培养液EGM-2培养,种植于提前包埋了纤维连接蛋白的培养皿进行体外扩增,分别取48 h内贴壁细胞和48 h后贴壁细胞,应用免疫组化和免疫荧光技术鉴定内皮细胞系列标志:CD34、CD31、FLK-1、VE-Cadherin和Ⅷ因子以及祖细胞标志CD133。结果经过梯度密度离心和贴壁法选择的细胞表达内皮细胞特异性抗原CD34、CD31、FLK-1、VE-Cadherin和Ⅷ因子,部分表达CD133。培养至第5代细胞形态基本相似,细胞总数可以达到108以上。结论自小儿骨髓可以分离培养出内皮细胞和内皮祖细胞并能体外扩增,可以作为构建组织工程血管、补片或带瓣管道的种子细胞来源。

Objective To test whether endothelial cells and their progenitor cells could be isolated from children＇s bone marrow and whether they could be cultured and proliferated in vitro. Methods Mononuclear cells were isolated from bone marrow of the children with congenital heart disease and cultured in endothelial growth medium-2 supplied with recombined human VEGF, IGF, b-FGF, FBS and ascorbic acid. Discs were coated with fibronectin before culture. Immunocytochemistry and immunofluorescence were performed to analyze the expression of factor VIH, CD31, CD34, FLK-1, VE-Cadherin, and progentitor cell marker CD133. Results Cultured cells were positively stained for factor VIH, CD31, CD34, FLK-1 and VE-Cadherin. They partly expressed CD133. Its number was more than 10s when cultured through 5 passages. Conclusion Human endothelial cells or their progenitor cells can be isolated from bone marrow. Its number can reach the seeding cell criteria required in vessel engineering when cultured in vitro. They could be used as an auto-donated seeding cell resource in vessel or valve engineering.