天津医药

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差速贴壁法培养大鼠骨髓内皮祖细胞的实验研究

李辰运,陈剑秋   

  1. 天津医科大学第二医院
  • 收稿日期:2010-08-11 修回日期:2010-10-23 出版日期:2011-04-15 发布日期:2011-04-15
  • 通讯作者: 李辰运

Endothelial Progenitor Cells Derived from Rat Bone Marrow Culture with Differential Adhesion Method

  • Received:2010-08-11 Revised:2010-10-23 Published:2011-04-15 Online:2011-04-15

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摘要： 摘要 目的：探索一种经济可靠、稳定、高纯度内皮祖细胞的培养方法。方法：采用密度梯度离心法分离培养大鼠骨髓血管内皮祖细胞，于包被人纤连蛋白的25ml培养瓶中贴壁培养，收集48h后的未贴壁细胞培养至第7天，通过细胞摄取DiL-acLDL、结合FITC-UEA-1的荧光双染阳性率从功能角度鉴定细胞，并通过流式细胞术动态测定细胞表面抗原CD133、flk-1、VE-cadherin(CD144)及CD31进一步鉴定。结果：48h未贴壁细胞培养，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；细胞摄取DiI-LDL，结合FITC-UEA-1荧光双染阳性率为（87.0±6.0）%。流式细胞术检测细胞表面抗原CD133、flk-1、VE-cadherin和CD31阳性率分别为61.01%、8.55%、8.20%、6.18%。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。为下一步研究提供方法学参考。

关键词: 差速贴壁法, 大鼠, 单个核细胞, 内皮祖细胞

Abstract: Abstract Objective：Explore an economic and reliable, stable, high purity of endothelial progenitor cells in culture methods.Methods：Rat bone marrow-derived EPCs were separated by density gradient centrifugation and cultured，then plated on 25ml culture flask adherent culture with human fibronection, 48h no adherent cells were collected cultured for 7 days, and then identified by being observed the experirment of uptaking DiL-acLDL and combining FITC-UEA-1 by fluorescence microscope . Also Flow Cytometry was performed to analyze the expression of CD133、flk-1、VE-cadherin(CD144) and CD31 dynamically.Results：48h no adherent cells cultured,the cells were small and spindle or polygonal in shape.Large numbers of typical endothelial progenitor cells coloy-forming unites were found. Cells DiI-acLDL uptake and FITC-UEA-1 combining fluorescent staining positive rate（87.0±6.0）%.Flow cytometry to detect cell surface antigen CD133, flk-1, VE-cadherin and CD31 positive rates were 61.01%, 8.55%,8.20%,6.18%. Conclusions：Differential attachment method is a reliable, stable, high purity of endothelial progenitor cells culture methods. Provide methodological references for further research.

Key words: Differential attachment method, Rat, Mononuclear cells, Endothelial progenitor cells