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目的：研究人血管内皮细胞在体外长期传代培养的方法。方法：采用０．１２５％胰酶ＥＤＴＡ液灌注冲洗法，从人脐静脉中分离出血管内皮细胞（ＨＵＶＥＣ）；用０．０２％ＥＤＴＡ消化传代ＥＣ，用新生小牛视网膜提取物（ＲＤＳ）作为生长促进剂。应用光镜、电镜、免疫组化等方法对原代和继代内皮细胞进行鉴定和生长特性观察。结果：人脐静脉内皮细胞继代培养３６代。原代和继代内皮细胞胞浆中含有ＷＰ小体和Ⅷ因子相关抗原；传代ＥＣ保留原代ＥＣ的生物学特征。５０％血清明胶和大白鼠尾胶原预先覆膜培养板，提高ＥＣ贴壁率。结论：传代ＥＣ培养能代替原代ＥＣ培养作为体外研究模型。ＲＤＳ能维持ＥＣ长期传代培养。
Objective: To study the method of long-term subculture of human vascular endothelial cells in vitro. Methods: HUVECs were isolated from human umbilical vein by 0.125% trypsin EDTA solution perfusion method. The passage ECs were digested with 0.02% EDTA, and neonatal calf retinal extracts (RDS) were used as Growth promoter. Application of light microscopy, electron microscopy, immunohistochemistry and other methods of primary and secondary endothelial cell identification and growth characteristics observed. Results: Human umbilical vein endothelial cells were subcultured for 36 passages. Primary and secondary endothelial cells contain cytoplasmic W P bodies and Ⅷ factor-related antigen; subculture EC to retain the biological characteristics of primary EC. 50% serum gelatin and rat tail collagen pre-coated culture plates to improve EC adherent rate. CONCLUSION: Subculture EC can replace primary EC culture as an in vitro model. RDS can maintain EC long-term subculture.