人脐静脉内皮细胞的原代培养和鉴定

目的：探索建立稳定有效的脐静脉内皮细胞体外培养方法。方法：用0．1％II型胶原酶消化分离人脐静脉肉皮细胞，加入含内皮细胞生长因子的M199完全培养液中培养，用胰蛋白酶-EDTA进行消化传代培养，用光镜和免疫组化方法对培养的细胞进行形态观察和鉴定。结果：原代培养的内皮细胞在接种后24h后完全贴壁生长，第445天后融合呈铺路石样镶嵌排列，免疫组化可见胞浆中第Ⅷ因子相关抗原呈阳性反应，证实培养的细胞为内皮细胞。结论：脐静脉灌注II型胶原酶消化法配合M199完全培养液可获得高纯度的内皮细胞，细胞可传代5—6次，但细胞产出量不高，不能传10代以上，5代以后细胞形态变化较大，对于复杂的基础研究应用受限。

Primary Culture and Identification of Human Umbilical Vein Endothelial Cells

Objective： To investigate a stable way to culture the human umbilical vein endothelial cells（HUVECs）. Methods： HU- VECs were obtained from human umbilical vein, digested with 0.1% coUagenase type II and cultivated in M 199 culture medium containing 20μg/mL ECGF. Subculture was attained with trypsin/EDTA solution when cells were 90% confluent. The cells were detected under a fluorescence microscope with immunohistochemical method. Results： The primary cells could adhere to the plate completely within 24 hours and grow to a monolayer in 4-5 days. Confluent cells exhibited the characteristic ＂cobblestone＂ morphology in culture and were positively immunostained with antibodies against von Willeband factor（vWF）, which proved that the cultured cells were endothetial cells. Conclusion： Highly purified primary HUVECs can be acquired by digestion of collagenase type II and cultivation of M199 complete culture medium. Further subculture beyond passage 5 is not advisable since the cell growth rate may decline and phenotypic changes become evident which may limit its application in complicated basic research.