人脐静脉内皮细胞体外培养方法

The cultivation of the human umbilical vein endothelial cell in vitro

目的探索人脐静脉内皮细胞体外培养方法和注意事项。方法采用0.1%Ⅰ型胶原酶分离脐静脉内皮细胞，于RPMI-1640和20%FBS内培养，待细胞80%融合后，以0.25%胰蛋白酶和0.02%EDTA消化传代，并以倒置光显微镜观察内皮细胞生长情况，以免疫组化方法对内皮细胞进行鉴定。结果原代培养细胞在接种4 h后开始贴壁生长，5-7 d后融合成单层，倒置光显微镜下观察细胞呈鹅卵石状排列，有接触抑制现象，免疫组化显示细胞胞浆内Ⅷ因子相关抗原阳性。结论用胶原酶灌注消化脐静脉是获得内皮细胞的一种较可靠的方法，成活率较高，培养的多个环节均需关注。

Objective To explore the culture method of the human umbilical vein endothelial cell （HUVEC） in vitro. Methods HUVECs were acquired with type Ⅰ collagenase treatment and cultivated in RPMI-1640 conraining 20% FBS; the passage was developed with 0.25% trypsinase and 0.02% EDTA. The ECs were identified under the light microscope with immunohistochemical method. Results The primary cells began to attach and grow after being inoculated 4 hours, and grew to monolayer 5 - 7 days later. They presented cobblestone-appearance with contact inhibition under the light microscope, and the antigens of human Ⅷ factor were posirive according to immunohistochemical method, Conclusion Collagenase type Ⅰ perfusion is an effective way to cultivate the human umbilical vein endothelial cells in vitro.