关键词: 内皮祖细胞, 细胞培养, 外周血, 表面抗原, 流式细胞仪

Abstract:

BACKGROUND: Although peripheral blood in adults can provide abundant source of cells, the content of endothelial progenitor cells is low. It is necessary to establish a stable and mature in vitro cultivation system of endothelial progenitor cells in order to achieve a better application in tissue engineering and cell therapy.
OBJECTIVE: To establish a stable cultivation system of endothelial progenitor cells from human peripheral blood in vitro, including cell isolation, culture and expansion.
METHODS: Mononuclear cells were isolated from the human peripheral anticoagulant blood sample by density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. The cells were cultured in endothelial progenitor medium. Non-adherent cells were washed away. The adherent cells were collected at the 6th day after cultivation. The morphologic changes of endothelial progenitor cells were observed by hematoxylin-eosin staining through invert microscope. Growth curves of cells of passages 1 and 3 were measured by MTT and cell counting. The cultured cells were identified by flow cytometry based on the presence of specific surface markers in progenitor cells and endothelial cell line.
RESULTS AND CONCLUSION: According to the growth curves, cells entered logarthmic growth phase at the 3rd day after inoculation, and then entered platform phase at the 6th day after inoculation. Cell proliferation rate slowed with increasing passage times. The stem cell surface markers CD34, CD133 and endothelial cell surface markers von willebrand factor and vascular endothelial growth factor receptor 2 were expressed at the same time. These results demonstrate that endothelial progenitor cells derived from human peripheral blood can be propagated in vitro.