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差速贴壁分离法：获得小鼠骨髓内皮祖细胞诱导分化培养的最佳贴壁时间

杨娜娜, 焦鹏, 李大伟, 王孟赞, 姚树桐, 宗传龙, 秦树存^*

泰山医学院动脉粥样硬化研究所，泰安 271000；泰山医学院附属聊城市人民医院心血管内科，聊城 252004

摘要

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1 d贴壁细胞组，取1 d非贴壁细胞再接种为3 d贴壁细胞组，继续培养2 d取非贴壁细胞再接种为3 d非贴壁细胞组，继续培养20 d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，1 d贴壁细胞中CD34+、FLK-1+、CD34+/FLK-1+细胞数明显多于3 d贴壁细胞和3 d非贴壁细胞(P < 0.01)；1 d贴壁细胞粘附能力、成血管能力均显著高于3 d贴壁细胞和3 d非贴壁细胞；1 d贴壁细胞和3 d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3 d非贴壁细胞(P < 0.01)；3 d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1 d贴壁细胞和3 d非贴壁细胞(P < 0.01)。以上结果提示，小鼠骨髓单个核细胞1 d贴壁诱导分化的内皮祖细胞生物学功能显著高于3 d贴壁细胞和3 d非贴壁细胞；3 d贴壁细胞较1 d贴壁细胞和3 d非贴壁细胞表达较多的VEGF，1 d贴壁细胞和3 d贴壁细胞eNOS表达高于3 d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1~3 d。

关键词： 内皮祖细胞; 差速贴壁; 小鼠; CD133; FLK-1

分类号：R34

[Differential time attachment: optimization of the adherent time to obtain mouse bone marrow-derived endothelial progenitor cells.] [Article in Chinese]

YANG Na-Na, JIAO Peng, LI Da-Wei, WANG Meng-Zan, YAO Shu-Tong, ZONG Chuan-Long, QIN Shu-Cun^*

Institute of Atherosclerosis, Taishan Medical University, Taian 271000, China； Institute of Cardiology, Liaocheng Hospital, Taishan Medical University, Liaocheng 252004, China

Abstract

The different biological functions were studied in mouse bone marrow-derived endothelial progenitor cells isolated by differential time attachment to obtain the optimal adherent time in this study. Density gradient centrifugation-isolated bone marrow mononuclear cells were seeded on the fibronectin-coated dish. The 1-day cultured unattached cells were seeded on the second dish for 2 more days. Then unattached cells in the second dish were seeded on the third dish. The cells on 3 dishes were defined as 1-day adherent cells, 3-day adherent cells and 3-day unattached cells, respectively. After 20-day culture, the biological functions, such as the percentage of biomarkers, the ability of adhesion, and the ability of forming tubes in vitro were analyzed. The results showed that the percentages of positive CD34, FLK-1, and CD34/FLK-1 expressions in 1-day attached cells were significantly increased compared to those in the 3-day adherent or unattached cells (P < 0.01), which showed the strongest adhesion ability. The expression of eNOS in 1- or 3-day adherent cells was significantly higher than that in 3-day unattached cells (P < 0.01). The expression of VEGF in 3-day adherent cells was significantly higher than that in 1-day adherent cells or 3-day unattached cells (P < 0.01). These results suggest the biological functions of 1-day adherent cells are significantly stronger than that of 3-day adherent or unattached cells. VEGF expression in 3-day adherent cells is higher than that in 1-day adherent cells or 3-day unattached cells. The expression of eNOS in 1-day adherent cells or 3-day adherent cells is higher than that in 3-day unattached cells. The optimal adherent time to obtain mouse bone marrow-derived endothelial progenitor cells is 1–3 d.

Key words: endothelial progenitor cell; differential attachment; mouse; CD133; FLK-1

收稿日期：2011-05-09　　录用日期：2011-07-21

通讯作者：秦树存　　E-mail: shucunqin@hotmail.com

引用本文：

杨娜娜, 焦鹏, 李大伟, 王孟赞, 姚树桐, 宗传龙, 秦树存. 差速贴壁分离法：获得小鼠骨髓内皮祖细胞诱导分化培养的最佳贴壁时间[J]. 生理学报 2011; 63 (6): 574-580.

YANG Na-Na, JIAO Peng, LI Da-Wei, WANG Meng-Zan, YAO Shu-Tong, ZONG Chuan-Long, QIN Shu-Cun. [Differential time attachment: optimization of the adherent time to obtain mouse bone marrow-derived endothelial progenitor cells.] [Article in Chinese]. Acta Physiol Sin 2011; 63 (6): 574-580 (in Chinese with English abstract).