先天性心脏病患儿骨髓单核细胞分离培养内皮祖细胞的实验研究

Experimental study on isolation,culture of endothelial progenitors from bone marrow mononeuclear cells in children with congenital heart disease

目的探索自先天性心脏病患儿骨髓单核细胞中分离培养内皮祖细胞的方法,期望为儿童组织工程血管、补片或带瓣管道的制备找到新的种子细胞来源。方法采集先天性心脏病患儿骨髓,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,取48h后贴壁细胞,应用免疫组织化学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。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。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。结论自先天性心脏病患儿骨髓单核细胞中可以分离培养出内皮祖细胞并能体外扩增,可以作为先天性心脏病患儿构建组织工程血管的种子细胞来源。

Objective To study the technique of isolation culture of endothelial progenitors from bone marrow mononeuclear cells in children with congenital heart disease in order to find out the novel resources of progenitor cells for preparing the tissue engineering vessel,patch and pedicular conduit for children. Methods Total mononuclear cells were isolated from bone marrow from children with congenital heart disease and cultured in endothelial growth medium-2 supplied with recombined human VEGF,IGF,b-FGF,FBS and ascorbic acid. Culture discs were coated with fibronectin before culture. Immunocytochemistry and immunofluorescence technique were performed to assay the endothelial cell markers ：factors 8, CD31,CD34,FLK-1 and ve-Cadherin. Results Cultured cells were positively stained for factor 8,CD31 ,CD34,FLK-1 and ve-Cadherin.The total number of proliferated cells was more than 10^8 with similar morphology, when cultured 5 passages. Conclusion Endothelial cells can be isolated from bone marrow in children with congenital heart disease. The number of proliferated cells cultured in vitro can reach the seeding cell criteria required in tissue engineering vessel. It could be used as an auto-donated seeding cell resource in tissue engineering for vessel or valve.