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本文旨在比较差速贴壁方法分离的不同时间点贴壁的小鼠骨髓单个核细胞诱导分化为内皮祖细胞的生物学特性,探讨最适宜贴壁时间。Ficoll密度梯度离心分离小鼠骨髓单个核细胞,接种于预先铺有纤维连接蛋白的培养板上,定义为1d贴壁细胞组,取1d非贴壁细胞再接种为3d贴壁细胞组,继续培养2d取非贴壁细胞再接种为3d非贴壁细胞组,继续培养20d后,分别检测3组诱导分化内皮祖细胞亚群表面标志、粘附能力和成血管能力。结果显示,1d贴壁细胞中CD34+、FLK-1+、CD34+/FLK-1+细胞数明显多于3d贴壁细胞和3d非贴壁细胞(P<0.01);1d贴壁细胞粘附能力、成血管能力均显著高于3d贴壁细胞和3d非贴壁细胞;1d贴壁细胞和3d贴壁细胞内皮型一氧化氮合酶(endothelial NO synthase,eNOS)表达量无显著差异,但均显著高于3d非贴壁细胞(P<0.01);3d贴壁细胞血管内皮生长因子(vascular endothelial growth factor,VEGF)表达水平显著高于1d贴壁细胞和3d非贴壁细胞(P<0.01)。以上结果提示,小鼠骨髓单个核细胞1d贴壁诱导分化的内皮祖细胞生物学功能显著高于3d贴壁细胞和3d非贴壁细胞;3d贴壁细胞较1d贴壁细胞和3d非贴壁细胞表达较多的VEGF,1d贴壁细胞和3d贴壁细胞eNOS表达高于3d非贴壁细胞。小鼠骨髓内皮祖细胞诱导分化培养最适宜贴壁时间为1～3d。
The purpose of this paper is to compare the biological characteristics of adherent mouse bone marrow mononuclear cells differentiated into endothelial progenitor cells at different time points separated by the differential adherence method and to explore the most suitable adherent time. Ficoll density gradient centrifugation mouse bone marrow mononuclear cells were seeded on fibronectin pre-plated plate, defined as 1d adherent cells group, take 1d non-adherent cells and then adherent to the 3d adherent cells, continue to culture 2d non-adherent cells re-seeded as 3d non-adherent cells, cultured for another 20d, were detected three groups of differentiated endothelial progenitor cells surface markers, adhesion and angiogenesis. The results showed that the number of CD34 +, FLK-1 +, CD34 + / FLK-1 + cells in adherent cells was significantly higher than that in 3d adherent cells and 3d non-adherent cells (P <0.01) The ability of angiogenesis was significantly higher than that of 3d adherent cells and 3 d non-adherent cells. There was no significant difference in the expression of endothelial nitric oxide synthase (eNOS) between adherent cells and adherent cells at 1 d (P <0.01). The expression of vascular endothelial growth factor (VEGF) in 3d adherent cells was significantly higher than that in 1d adherent cells and 3d non-adherent cells (P <0.01). The above results suggest that the biological function of mouse bone marrow mononuclear cells induced adherent differentiation on day 1 is significantly higher than that of 3d adherent cells and 3d non adherent cells; 3d adherent cells are more adherent than 1d adherent cells and 3d adherent The higher expression of VEGF, 1d adherent cells and 3d adherent cells eNOS expression than 3d non-adherent cells. Mouse bone marrow endothelial progenitor cells induced differentiation of the most suitable adherent time is 1 ~ 3d.