正常成人粒细胞集落刺激因子动员外周血内皮祖细胞的生物学特性

摘要

BACKGROUND:Endothelial progenitor cells have good prospects for clinical application;especial y as seed cells, they are involved in construction of tissue engineered blood vessels and disease treatment. OBJECTIVE:To investigate the biological characteristics of endothelial progenitor cells derived from granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood. METHODS:Mononuclear cells from G-CSF mobilized peripheral blood (n=9) and normal adult peripheral blood (n=8) were obtained and cultured in expanded medium. The immunophenotype of endothelial progenitor cells was investigated by FACS and immunohistochemistry. Real-time PCR was used to detect the expression of different cytokines. Proliferation and adhesion ability of endothelial progenitor cells were detected by MTT assay and adhesion assay. Moreover, the abilities of vasculogenesis in vitro and Dil-labeled acetylated low density lipoprotein uptake were detected. The acquired cells were seeded onto the basilar membrane gel containing vascular endothelial growth factor and basic fibroblast growth factor to induce angiogenesis. RESULTS AND CONCLUSION:Endothelial progenitor cells derived from G-CSF mobilized peripheral blood were similar to those from normal adult peripheral blood in phenotype and morphology. FACSs and immunohistochemistry showed that endothelial progenitor cells were positive for endothelial cellmarkers, such as CD31, vWF, CD34, FLK-1, VE-Cadherin and CD133. In addition, the expression of vascular endothelial growth factor and stromal cel-derived factor 1 were significantly increased in G-CSF mobilized endothelial progenitor cells. Moreover, endothelial progenitor cells derived from two sources possessed the abilities of angiogenesis in vitro and Dil-labeled acetylated low density lipoprotein uptake. But, the number and the proliferation ability of endothelial progenitor cells derived from G-CSF mobilized peripheral blood were better than that of endothelial progenitor cells derived from normal adult peripheral blood. These findings indicate that there is a population of cells with characteristics of endothelial progenitor cells in G-CSF mobilized peripheral blood. They possess the abilities of vasculogenesis in vitro. Moreover, compared to endothelial progenitor cells derived from normal adult peripheral blood, we could obtain more endothelial progenitor cells in G-CSF mobilized peripheral blood and those cells show better proliferation ability.%背景：内皮祖细胞具有很好的临床应用前景，特别是作为种子细胞参与组织工程血管构建及疾病治疗。目的：观察粒细胞集落刺激因子动员的外周血中内皮祖细胞的生物学特性。方法：获取粒细胞集落刺激因子动员正常人的外周血单个核细胞，同时获取8例正常成人外周血单个核细胞作为对照。将获取的细胞接种在预铺纤维连接蛋白的培养皿中进行体外培养。FACS和免疫组化法检测获取细胞的免疫表型；采用 MTT 比色法和黏附能力测定实验检测内皮祖细胞的增殖和黏附能力；实时定量PCR检测血管形成相关细胞因子的表达；荧光标记的乙酰化低密度脂蛋白(Dil-acLDL)吞噬实验鉴定内皮功能；将获取的细胞接种在含有血管内皮细胞生长因子和碱性成纤维细胞生长因子的基底膜胶中诱导血管生成。结果与结论：动员后外周血和未动员外周血内皮祖细胞具有相似的细胞形态和免疫表型，FACS 和免疫组化检测结果显示上述两种内皮祖细胞都表达内皮细胞特异性抗原 CD34、CD31、FLK-1、ve-Cadherin、vWF和CD133。内皮细胞生长因子和基质细胞衍生因子1在动员后外周血内皮祖细胞中的表达水平增加；两种来源的内皮祖细胞都具有吞噬 Dil-acLDL 和在体外诱导血管生成的能力。但是，动员的外周血中内皮祖细胞的数量和增殖能力明显强于未动员外周血中的内皮祖细胞。结果可见动员后的外周血中可以分离出具有内皮祖细胞特性的细胞群体，该群体具有体外诱导血管生成的能力；与未动员的外周血内皮祖细胞相比，动员后外周血内皮祖细胞数量明显增加并具有更好的增殖能力。