摘要
目的:建立能大量分离人脐静脉内皮细胞的体外培养的简便方法,分析人脐静脉内皮细胞抗原表达的特点。方法:采用玻璃接管连接医用三通管灌注0.133%胶原酶I法分离脐静脉内皮细胞,培养于10%胎牛血清的人内皮细胞培养液(含β-促内皮细胞生长因子和肝素钠),培养皿用1.5%明胶包被,促进内皮细胞贴壁。培养过程中观察细胞的形态特征,同时行免疫组织化学染色检测第八因子相关抗原、CD31表达情况,鉴定内皮细胞来源。结果:成功获取人脐静脉内皮细胞,原代人脐内皮细胞24h贴壁,第5日细胞融合;第八因子相关抗原和CD31呈广泛的阳性表达,第八因子相关抗原阳性染色程度高于CD31,证实为人脐静脉内皮细胞。结论:应用玻璃接管连接医用三通管灌注胶原酶消化法分离脐静脉内皮细胞,利用10%优质胎牛血清的人内皮细胞培养基,添加β-促内皮细胞生长因子和肝素钠,并用1.5%明胶包被培养皿,能简单有效培养人脐静脉内皮细胞。
Purpose：To establish a simple and convenient method for the culture of human umbilical vein endothelial cells （HUVECs） and study its characterization in vitro. Methods： Human umbilical cord was isolated and digested by collagenase type I. And then,it was cultured in 1.5% geltain coated dish with 10% fetal bovine serum（FBS）,heparin sodium and β-endothelial cell growth factor（β-ECGF） in human endothelial basal growth medium.HUVECs were identified by anti-human factor Ⅷ related antigen and CD31 immunohistochemical staining. Results： We can successfully culture HUVECs in this way. HUVECs attached the dish in 24 hours and the confluence was seen in 5 days. HUVECs were generally positive for anti-human factor Ⅷ related antigen and CD31. Conclusion： It is a fast and effective method to culture purified HUVECs successfully in which we use collagenase type I to digest HUVECs with glass tube connected to T-tube,human endothelial basal growth medium containing 10% FBS and heparin sodium and β-ECGF in 1.5% geltain coated dish.
关键词
人脐静脉内皮细胞
细胞培养方法
鉴定
Human Umbilical Vein Endothelial Cells
Culture
Identification
