小鼠骨髓源性内皮祖细胞的体外培养及标志物鉴定

［摘要］目的: 探讨小鼠骨髓来源的血管内皮祖细胞（endothelial progenitor cells，EPCs）的体外培养、诱导分化及表面标志物的鉴别。方法： 收集ICR小鼠骨髓EPCs，Ficoll分离单核细胞，使用含有血管内皮生长因子（VEGF）、成纤维细胞生长因子(FGF)的培养基培养。分别在细胞培养5，10，15及20 d后检测细胞表面的内皮细胞标志物。结果： 培养5 d后细胞开始出现上皮细胞的形态，有伪足出现；培养10 d，细胞开始增殖，成圆形，出现梭形细胞；培养15 d，细胞出现较为典型的内皮细胞形态，铺路石状；培养20 d，细胞出现长梭形拉网状，即类似成熟内皮细胞形态。免疫荧光染色及流式定量检测结果显示，CD34在EPCs中的表达随培养时间延长有渐变减弱的趋势；血管内皮生长因子受体 2（VEGFR-2）在EPCs的表达随培养时间延长有渐变增强的趋势。结论： 骨髓EPCs在特定诱导条件下可分化出典型的成熟内皮细胞，取材方便，扩增能力较强。

Culture and identification of mouse bone marrow-derived endothelial progenitor cells in vitro

Objective： To develope methods of in vitro culture,differentiation and identification of mouse bone marrow derived-endothelial progenitor cells（EPCs）.Methods： Collected EPCs from ICR mouse,differentiated mononuclear cells by density gradient centrifugation,cultured in medium with vascular endothelial growth factor（VEGF）,fibroblast growth factor（FGF） and detected the cell surface markers of endothelial cells after 5 days,10 days,15 days,20 days.Results： Cells began to appear epithelial cell morphology after 5 days,and pseudopods appeared;after 10 days of culture,cells began to proliferate,turned round and spindle cells began to appear;after 15 days,cells showed typical morphology of endothelial cells,formed cobblestone like structure,after 20 days,cells performed like long fusiform pulling nets,just similar with mature endothelial cell morphology.Immunofluorescence stain and flow quantitative test results showed,CD34 expression level in EPCs with the extension of incubation time had a gradual weakening trend;VEGFR-2 expression in EPCs with the incubation time had a gradual increasing trend.Conclusions： EPCs from bone marrow cultured in specific conditions could be induced differentiate typical of mature ECs;it was convenient and had strong amplification ability.