胰蛋白酶消化法体外培养和鉴定人脐静脉血管内皮细胞

背景:体外建立稳定可靠的人脐静脉血管内皮细胞模型,目前培养方法缺乏统一的标准。目的:探索脐静脉内皮细胞的体外培养的方法。方法:采用2.5g/L胰蛋白酶和0.02%EDTA消化、分离、体外原代培养及消化传代脐静脉内皮细胞,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,根据细胞特有的形态学特征和Ⅷ因子进行内皮细胞的鉴定。结果与结论:种植在培养瓶中的内皮细胞2h贴壁生长,24h换液后内皮细胞80%融合,细胞状态好,内皮细胞呈单层铺路石样外观,经过镜下观察和Ⅷ因子相关抗原鉴定证明是脐静脉内皮细胞。证实用胰蛋白酶灌注脐静脉是一种简单、实用的获得人脐静脉血管内皮细胞的方法,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。

Culture and identification of human umbilical vein endothelial cells in vitro using Trypsin digestion method

BACKGROUND:The establishment of human umbilical vein endothelial cells model in vitro has significant meaning for the study of neovascularization,but human umbilical vein endothelial cells are difficult to cultivate in vitro and there is not a criterion.OBJECTIVE:To explore how to harvest and identify human umbilical vein endothelial cells in vitro.METHODS:0.25 g/L Trypsin and 0.02% ethylene diamine tetraacetic acid perfusion method was used to isolate human umbilical vein endothelial cells from the fresh umbilical cord,and the cells were cultured and amplified.The composition of culture medium was simplified by not adding vascular endothelial cell growth factor and heparin.Human umbilical vein endothelial cells grown to 80% confluence were identified by morphological observation and immunofluorescence method.RESULTS AND CONCLUSION:The endothelial cells spread on the bottom of the dishes within 2 hours,then coalesced and grew to form a confluent monolayer of polygonal cells within 24 hours.The cultured cells were identified as human umbilical vein endothelial cells.Trypsin perfusion is a simple and effective method for collection of human umbilical vein endothelial cells.Cells harvested with this protocol can be used as models on research of vascular endothelial cells in vitro.