Biological characteristics of endothelial progenitor cells derived from granulocyte colony-stimulating factor mobilized peripheral blood

doi:10.3969/j.issn.2095-4344.2014.32.018

Abstract

Abstract:

BACKGROUND: Endothelial progenitor cells have good prospects for clinical application; especially 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 cell markers, such as CD31, vWF, CD34, FLK-1, VE-Cadherin and CD133. In addition, the expression of vascular endothelial growth factor and stromal cell-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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: endothelial cells, granulocyte colony-stimulating factor, hematopoietic stem cell mobilization

CLC Number:

R394.2

Bai Li-ping, Zhao Zhi-hong, Chen Chong, Wang Jin-huan, Wang Zhen-ling, Zhao Zhi-gang. Biological characteristics of endothelial progenitor cells derived from granulocyte colony-stimulating factor mobilized peripheral blood[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(32): 5190-5196.

Figures/Tables 2

2.1 内皮祖细胞的鉴定和形态学特点于倒置显微镜下观察，原代培养的贴壁细胞形态表现为梭形或多边形，胞浆丰富，核仁明显，呈不规则排列生长。传代后细胞形态不发生改变，当细胞融合后呈“铺路卵石样”。通过共聚焦显微镜观察，UEA-Ⅰ和DiLDL双染色阳性的细胞被认为是正在分化的内皮祖细胞，实验从粒细胞集落刺激因子动员的外周血和对照组(正常成人外周血)中都培养出内皮祖细胞(图1)。通过对原代培养细胞的计数结果显示粒细胞集落刺激因子动员的外周血中内皮祖细胞的数量[(102.0±2.6)/视野(×200)]明显高于正常成人外周血中的内皮祖细胞[(59.0± 5.1)/视野(×200)，P < 0.01]。粒细胞集落刺激因子动员的外周血中获取的内皮祖细胞可以在体外稳定培养6-8代，随后细胞增殖减慢，细胞形态变大，胞浆中颗粒增多，呈现老化状态；而正常成人外周血中的内皮祖细胞最多在体外培养5代，然后细胞呈现老化状态。 2.2 内皮祖细胞的免疫表型通过流式细胞仪检测内皮祖细胞的免疫表型，发现原代获取的内皮祖细胞表达CD31、FLK-1、VE-Cadherin、vWF和CD133。随着细胞传代，CD31和vWF的表达逐渐增加，而CD133的表达下降，从第3代起，已经检测不出CD133的表达。免疫组化法检测结果也显示获取的内皮祖细胞表达CD31、FLK-1、VE-Cadherin和vWF(图2)。粒细胞集落刺激因子动员的外周血和对照组(正常成人外周血)中获取的内皮祖细胞具有相似的免疫表型。 2.3 内皮祖细胞的增殖和黏附能力采用MTT比色法检测了不同来源外周血内皮祖细胞的增殖能力，结果显示粒细胞集落刺激因子动员的外周血中获取的内皮祖细胞增殖能力明显强于正常成人外周血内皮祖细胞的增殖能力(0.84±0.03 vs. 0.47±0.04，P < 0.05)。另外，还检测了不同来源外周血内皮祖细胞的黏附能力，结果显示粒细胞集落刺激因子动员的外周血中获取的内皮祖细胞，与正常成人外周血内皮祖细胞具有相似的黏附能力[(96.32±5.61)/视野(×200) vs. (104.12±2.68)/视野(×200)，P > 0.05]。"

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