人脐静脉内皮细胞的长期传代培养

Long-term subculture of the endothelial cells of human umbilical cord vein in vitro

本实验在纤维连接蛋白(Fibronectin,Fn)预处理的塑料培养皿上,用199培养基(内含20％小牛血清)。成功地将人脐静脉内皮细胞以1:2-3比率传代培养两个月,传15代。培养细胞具有第Ⅷ因子相关抗原;胞质中存在Weible-Palade小体;细胞倍增时间为24h;培养4—6天,细胞单层融合,复苏成功率为70％左右;染色体检查2n=46。培养50天内,条件培养液中均检测到Von Willebrand因子和6-keto-PGF。

The endothelial cells of human umbilical cord vein (HUVEC) were grown for 15 passages for 2 monthes at a split ratio of 1 = 2-3 on plastic culture dishes which were pretreated with human fibronectin ( HFn ) matrix in medium 199 supplemented with 20% calf serum and endothelial cell growth supplement ( ECGS ) which was prepared in our laboratory. Histopogical and cytobiological investigations were performed in our experiment .The results are as follows : Cultured endothelial cells of the human umbilical cord vein were FⅧR; Ag positive cells.Weible-palade bodies in the cytoplasm were observed with transmission electron microscope. Cells exhibited a population doubling time of 24 hours and grew to confluence in 4-6 days. Formation of a contact-inhibited cell monolayer then followed. The cells were preserved in liquid nitrogen for one month, their resuscitative survival rate was about 70%. Chromosomal number was 46. During the course of a 50-day culture, both von willebrand factor and 6-keto-PGF 1a wore detected in the culture medium.