关键词: 内皮细胞, 细胞培养, 鉴定, 酶消化, 血管组织工程

Abstract:

BACKGROUND: Although the methods of mechanical scrapping, tissue block transplantation, and enzyme digestion to culture endothelial cells have been quite mature, it has been constantly updated.
OBJECTIVE: To explore the culture and identification of rabbit aortic endothelial cells.
METHODS: Aorta was derived from one-week-old New Zealand rabbit. Following removing the adventitia, the endothelium was digested in the mixture of 2 g/L type Ⅰ collagen, 2 g/L type Ⅱ collagen, 2 g/L type Ⅳ collagen, and 2 g/L type Ⅴ collagen (1:1:1:1) for 20 minutes, and the digestion was stopped with culture medium (1:1). Cells in the endothelium were lightly scraped to make cell suspension, which was centrifuged to precipitate cells with DMEM culture medium containing 20% fetal bovine serum, 1 μg/L VEGF, 2 μg/L bFGF, and 6 U/L gentamicin. The cells were then blown into single cells and the culture liquid was changed every 48 hours. The cells were passaged according to the ratio of 1:2. Inverted phase contrast microscope was used to observe cell culture; immunohistochemistry and immunofluorescence were used to determine Ⅷ-related antigen; electron microscope was used to detect Weibel-Paladed body.
RESULTS AND CONCLUSION: VIII-related antigen and electron microscopy confirmed that the primary and 5-passage endothelial cells were successfully cultured, suggesting that rabbit aortic endothelial cells were cultured into cell line. VIII-related antigen and electron microscopy determined that a combination with W-P body was a good method to determine endothelial cells.