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目的探索人脐静脉血内皮祖细胞的分离培养,为内皮祖细胞的临床应用提供实验方法。方法选择脐静脉血,应用密度梯度离心法,获取单个核细胞,接种于预先包埋了人纤维连接蛋白的培养板,用加入生长因子VEGF165和bFGF的内皮细胞专用培养基EGM-2MV培养细胞,3d后,洗掉非贴壁细胞,换培养液继续培养至7d,收集贴壁细胞进行细胞分析。激光共聚焦显微镜进行细胞功能学鉴定,流式细胞术测定祖细胞和内皮细胞系标志,MTT比色法检测细胞的生长状态。结果经过梯度密度离心和贴壁法选择的细胞能特异性吸附FITC标记的荆豆凝集素并内吞DiI-acLDL,祖细胞标志CD133及内皮细胞特异性抗原CD34、KDR检测,其阳性率分别为(27.05±2.94)%、(16.37±2.69)%和(56.67±7.29)%;体外培养的内皮祖细胞具有良好的细胞增殖活性。结论人脐静脉血中可以分离培养内皮祖细胞,为内皮祖细胞的进一步研究及临床应用奠定了基础。
Objective To explore the isolation and culture of human umbilical vein endothelial progenitor cells and to provide experimental methods for the clinical application of endothelial progenitor cells. Methods Umbilical cord blood was collected. Mononuclear cells were obtained by density gradient centrifugation. The cells were inoculated into pre-embedded human fibronectin-plated plates. Cells were cultured with EGM-2MV, an endothelial cell-specific medium containing growth factors VEGF165 and bFGF, After 3 days, the non-adherent cells were washed off, the culture solution was changed to continue for 7 days, and the adherent cells were collected for cell analysis. Laser confocal microscopy was used to identify the cell function. Flow cytometry was used to detect the markers of progenitor cells and endothelial cells. MTT assay was used to detect the cell growth status. Results The cells selected by gradient density centrifugation and adherence method could specifically adsorb FITC labeled Vitexin and endocytosis DiI-acLDL, progenitor cell marker CD133 and endothelial cell specific antigen CD34 and KDR, the positive rates were (27.05 ± 2.94)%, (16.37 ± 2.69)% and (56.67 ± 7.29)%, respectively. EPCs cultured in vitro had good cell proliferative activity. Conclusion Human endothelial progenitor cells can be isolated and cultured in human umbilical cord blood, which lays the foundation for further research and clinical application of endothelial progenitor cells.