大鼠骨髓内皮祖细胞的分离培养与鉴定

背景：内皮祖细胞参与血管新生,但其分离、培养、鉴定方法目前并不统一。 目的：探索大鼠骨髓内皮祖细胞的分离培养及鉴定方法。 方法：使用密度梯度离心法及差速贴壁法联合的方法培养内皮祖细胞,在倒置显微镜下观察细胞生长情况及形态变化,使用Dil标记的乙酰化低密度脂蛋白和FITC标记的荆豆凝集素1双荧光染色、流式细胞仪检测细胞表面抗原CD34、CD133表达情况。 结果与结论：培养前4 d细胞增殖不明显,第5~10天迅速增殖,并可见细胞集落及线状结构形成。培养第7天的内皮祖细胞具有吞噬Dil标记的乙酰化低密度脂蛋白和FITC标记的荆豆凝集素1的功能。流式细胞仪检测体外培养第10天的细胞,CD133+ 细胞占19.2%,CD34+ 细胞占28.7%,CD34+/CD133+ 细胞占19.1%。说明密度梯度离心法联合差速贴壁法可在体外有效分离培养大鼠骨髓内皮祖细胞。 关键词：内皮祖细胞;细胞分离;细胞培养;骨髓;细胞鉴定 doi:10.3969/j.issn.1673-8225.2012.10.006

Separation,culture and identification of rat bone marrow-derived endothelial progenitor cells

BACKGROUND: Endothelial progenitor cells (EPCs) are involved in angiogenesis, but its isolation, culture and identification methods are not uniform currently. OBJECTIVE: To explore the methods of separation, culture and identification of rat bone marrow-derived EPCs. METHODS: EPCs were cultured using density gradient centrifugation and differential velocity adherent methods, growth and morphology changes of cells were observed under inverted microscope. The expression of cell surface antigen CD34 and CD133 were detected by using Dil labeled acetylated low-density lipoprotein (Dil-ac-LDL) and FITC-labeled Ulex europaeus agglutinin 1 (FITC-UEA-1) double dyeing and flow cytometry.  RESULTS AND CONCLUSION: For the first four days, cell proliferation was not obvious, at the 5-10 days, cell proliferation was obvious, and colony-forming unit (CFU) and lineage structure could be seen. At the 7th day, EPCs could swallow the function of DiL-ac-LDL and FITC-UEA-1. At the 10th day, in vitro flow cytometry detection showed that the CD133+ cells were accounted for 19.2%, CD34+ cells were accounted for 28.7%, CD34+/CD133+cells were accounted for 19.1%. Using density gradient centrifugation and differential velocity adherent methods can separate and culture bone marrow derived EPCs.