一种改进的人脐静脉内皮细胞的培养方法  

An improved method of culturing human umbilical vein endothelial cells

作　　者：李琴山[1] 冯赞杰[1] 刘洋[1] 徐海燕[1] 钱民章[1] 

机构地区：[1]遵义医学院生化教研室,贵州遵义563000

出　　处：《第四军医大学学报》2007年第3期276-278,共3页Journal of the Fourth Military Medical University

基　　金：贵州省科学技术基金(2003-3015);遵义医学院硕士启动基金

摘　　要：目的：建立稳定的人脐静脉内皮细胞分离和培养的方法．方法：采用胰蛋白彰胶原酶（1：1）混合酶液消化法培养人脐静脉内皮细胞，简化完全培养液组分（不添加血管内皮细胞生长因子、肝素等辅助因子），当原代培养细胞80％以上汇合后用胰蛋白酶消化传代；用形态学、免疫细胞化学法进行鉴定．结果：种植在培养瓶中的内皮细胞4h贴壁生长，48～96h生长最快，7—10d汇合．内皮细胞呈单层铺路石样外观，Ⅷ因子相关抗原（Ⅷ：Ag）和内皮细胞生长因子受体2（KDR）鉴定内皮细胞纯度达95％以上．结论：用混合酶液灌注脐静脉消化内皮细胞是获取内皮细胞的一种好方法，可靠性大，成功率高，可以构建体外研究血管内皮细胞的模型．AIM： To establish a stable method to isolate and culture human umbilical vein endothelial cells （hUVEC） in vitro. METHODS： Endothelial cells of vein from newborn umbilical cord were successfully isolated using a combination of eollagenase and trypsin （ 1：1 ）. Cells were not digested with trypsin until the cells reached confluence over 80%. The composition of culture medium was simplified by not adding vascular endothelial cell growth factor and heparin, The cultured cells were verified as hUVEC by morphology and immunofluorescence staining. RESULTS： The endothelial cells initially spread on the bottom of the dishes in 4 h, then coalesced and grew to form confluent monolayers of polygonal cells within 7 - 10 d. The cultured cells had a cobblestone appearance with a strict monolayer growth and contact inhibition. The purity of endothelial cells was more than 95% identified by immunocytochemistry staining for Ⅷ：Ag and KDR. CONCLUSION ： Method of injection with mixed Enzyme solution is an optimized protocol which is reliable and of high achievement ratio. Cells harvested with this protocol can be used as models on research of vascular endothelial cells in vitro.

关 键 词：内皮细胞 脐静脉 细胞培养 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]