兔主动脉内皮细胞培养及鉴定:内皮细胞分离方式及培养条件的改良

背景:获取内皮细胞的方法有机械刮取、组织块移植和酶消化法3种.一直以来,内皮细胞的培养方法也在不断的更新.目的:探讨兔主动脉内皮细胞的培养和鉴定方法.方法:取1周龄新西兰大耳白兔主动脉,剥去外膜,内膜面向下铺入2 g/L Ⅰ型胶原酶、2 g/L Ⅲ型胶原酶,2 g/L Ⅳ型胶原酶和2 g/L Ⅴ型胶原酶混合消化液中(按1:1:1:1:1混合)消化20min,按1:1加入培养基以终止消化.轻轻刮下内膜层细胞,将细胞悬液离心,用DMEM培养液(含胎生血清20%、VEGF 1 μg/L、bFGF 2 μg/L,庆大霉素6 U/L)混匀沉淀细胞,吹打分散至单个细胞培养,48 h后用首次换液.再按1:2分瓶传代培养.采用倒置相差显微镜观察细胞培养结果.免疫组织化学及免疫荧光鉴定Ⅷ因子相关抗原.电镜观察Weibel-Paladed小体.结果与结论:体外获得并培养5代内皮细胞.Ⅷ因子相关抗原及电镜观察W-P小体均证实实验成功的培养了原代及传代内皮细胞.提示兔主动脉内皮细胞可从主动脉获得并通过培养成为细胞系,Ⅷ因子相关抗原及电镜观察W-P小体联合鉴定是确定内皮细胞的良好方法.

Culture and identification of rabbit aortic endothelial cells: Modified isolation and culture of endothelial cells

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 9/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 digestionwas stopped with culturemedium(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.