摘要
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2g/LⅠ型胶原酶、2g/LⅢ型胶原酶,2g/LⅣ型胶原酶和2g/LⅤ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(含胎牛血清20%、VEGF1μg/L、bFGF2μg/L,庆大霉素6U/L)混匀沉淀细胞,吹打分散至单个细胞培养,48h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。
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.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第11期1919-1922,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
