差速贴壁法体外培养人脐血内皮祖细胞的实验研究

Endothelial Progenitor Cells Derived from Human Umbilical Cord Blood Culture in Vitro with Differential Attachment Method

目的建立一种经济可靠、稳定、高纯度内皮祖细胞的培养方法。方法采用 Ficoll 密度梯度离心法从人脐血中分离单个核细胞,于包被人纤连蛋白的培养皿中贴壁培养,收集48 h后的悬浮细胞重新贴壁培养至第7天,利用免疫组化、免疫荧光及流式细胞术对培养的细胞进行鉴定。结果培养的细胞呈短梭形、多角型、胞体小;可见到大量典型的内皮祖细胞克隆;vWF 和 flk-1免疫组化细胞阳性率>95%,免疫荧光 Dil-ac-LDL 和 FITC-UEA-1双染阳性的细胞阳性率>98%,流式分析 CD_(133)^+细胞的百分率为(7.0±1.8)%。结论差速贴壁法是一种经济可靠、稳定、高纯度内皮祖细胞的培养方法。

Objective To establish a practical, stable and high purity endothelial progenitor cells culture method in vitro. Methods Human umbilical cord blood mononuclear cells were isolated by Ficoll density-gradient centrifugation, then plated on dishes coated with human fibronectin. After 48 hours, the nonaderent cells were collected and replated onto fibronectin-coated dishes. After 7 days of culture, the cells were identified with the techniques of immunohistochemistry, immunofluorescence and flow cytometer. Results The cultured cells were small and spindle or polygonal in shape. Large numbers of typical endothelial progenitor cell colony-forming units were found, vWF and Flk-1 proteins expression were identified in more than 95% of the attached cells with 98% of them showing positive Dil-ac-LDL and FITC-UEA-1. According to the results from fluorescence-activated cell sorting （FACS）, 7.0% ±1.8% of cells were recognized as CD133^＋. Conclusion Differential attachment technique is a practical and stable method for obtaining highly purified endothelial progenitor cells.