骨髓来源血管内皮祖细胞的体外分离培养和鉴定

Bone marrow-derived endothelial progenitor cells isolation and culture

目的:探索体外分离培养骨髓来源血管内皮祖细胞的方法。方法:将骨髓细胞接种至明胶涂层的培养皿内,用含10%胎牛血清、添加50μg/ml ECGS的M199培养液,置37℃、体积分数为5%的CO2饱和湿度恒温培养箱培养,0.05%胰酶-EDTA消化传代。通过CD31免疫荧光染色、荆豆凝集素免疫细胞化学染色及毛细血管腔形成能力检测进行鉴定。结果:培养5～7天,内皮祖细胞的早期克隆形成,2周后细胞表现出典型的"鹅卵石"状。可与荆豆凝集素特异性相结合;内皮细胞特异性表面标志CD31荧光染色呈阳性表达;培养过程中可形成管腔状结构。结论:自骨髓可以获取足量的EPCs。

Objective To isolate and culture endothelial progenitor cells（EPCs）.Methods Bone marrow cell were cultured in dishes coated with 1% gelatin with M199 medium supplemented with 10% fetal bovine serum and 50μg/mL endothelial cell growth supplements,at 37°C in a humidified atmosphere of 5% CO2.Subcultured using trypsin-EDTA.EPCs were identified by immunofluorescence staining for CD31 expression,ability of lectin binding.Further characterisation of EPCs was performed by analysing capillary tube formation.Results EPCs colonies appeared between 5 and 7 days of culture and defined as a central core of rounded cells.Two weeks later,cells presented the characteristic cobblestone morphology.The differentiation status of EPCs was confirmed by binding UEA-1 plant lectin and expressing CD31.Further,tubular network was formed.Conclusions When induced in culture,EPCs can be obtained,exhibit long term proliferative potential.