骨髓内皮祖细胞EPCs两种分离方法及其对比研究

Comparative study on two separating methods of bone marrow-derived endothelial progenitor cells in vitro

目的:研究免疫磁珠法和贴壁换液法分离骨髓内皮祖细胞的可行性和条件,并比较两种方法优缺点.方法:利用免疫磁珠法和贴壁换液法从骨髓中分别提取内皮祖细胞,体外观察细胞生长及形态变化,通过检测细胞血管内皮生长因子受体-2(VEGFR-2)、Ⅷ因子相关抗原抗体表达、摄取乙酰化-低密度脂蛋白(Ac-LDL)情况和超微结构等进行鉴定.同时,流式细胞检测分离所得细胞的纯度、细胞计数测定分离所得细胞的数量及MTT法检测细胞生长增殖状况等,对两种分离方法进行对比研究.结果:用免疫磁珠法和贴壁换液法从骨髓中分别提取的细胞,体外培养后细胞呈铺路石形,透射电镜显示细胞内具有特征性的W-P小体,免疫荧光染色检测细胞相关抗原VEGFR-2,Ⅷ/vWF呈阳性,同时细胞摄取Ac-LDL,证实为内皮祖细胞.免疫磁珠法与贴壁换液法相比,分离所得内皮祖细胞数量是后者2倍,纯度为76%,明显高于贴壁换液法的29%;同时,两者对细胞增殖生长无明显影响.结论:利用免疫磁珠法和贴壁换液法均可分离得到骨髓内皮祖细胞,它将成为新的组织工程种子细胞.免疫磁珠法分离得到细胞纯度更高、数量更大,但步骤复杂、价格昂贵;贴壁换液法得到细胞纯度和数量偏低,但方法简单、经济、实用性强.

AIM： To study the feasibility and conditions of immunity magnetic bean selection and attachment-changing culture methods to separate human bone marrow-derived endothelial progenitor cells（EPCs） in vitro, and to compare their traits of two separating methods. METHODS： First, we isolated EPCs from human bone marrow by magnetic bead selection and attachmentchanging culture methods, and then observed the growth and shape of the cells. EPCs were identified by test of uptake of acetylated low density lipoprotein （Ac-LDL）, ultrastructure observation by transmission electron microscope and immunofluorescence staining for the expressions of Ⅷ/vWF, vascular endothelial growth factor receptor-2 （VEGFR-2）. Two separating methods were compared in purification, amount and growth of EPCs. RESULTS： After induced by VEGF, bFGF and IGF-1, the cells purified from human bone marrow by magnetic bead selection or attachment-changing culture methods in vitro displayed cobblestone morphology and expressed characteristic Weibel-Paladle bodies under transmission electronic microscope. The cells were identified by uptake of Ac-LDL and the positive staining for VEGFR-2 and Ⅷ/vWF. Furthermore, the mount and purification of obtained EPCs （ 10 mL bone marrow） by immunity magnetic bean selection were 4. 5 × 10^5 and 76%, and those by attachmentchanging culture methods were 2 × 10^5 and 29%. The proliferation characteristics of EPCs obtained with two separating methods were similar. CONCLUSION： The EPCs can be purified by magnetic bead selection or attachment-changing culture methods from human bone marrow. It might be a new seed cell of tissue engineering. Compared with immunity magnetic bean selection,the mount and purification of EPCs obtained by attachment-changing culture methods is lower, but this method is more simple, convenient and practical.