人脐静脉内皮细胞原代培养及鉴定

摘要

Objective To establish a detailed protocol to cultivate and identify human umbilical endothelial cells.Methods HUVECs were harvested by type Ⅰ collagenase digestion at 37℃ and then cultured in ECM.Morphology of HUVECs was observed with a inverted phase-contrast microscope.The cellular marker-Ⅷ factor and CD3 1 were detected by immunofluorescence method.We detected endothelial isoform of nitric oxide synthase (eNOS)expression using western blotting.Results Highly pure HUVEC (95%) can be obtained by 0.2% type Ⅰ collagenase digestion.Under the inverted phase-contrast microscope, HUVECs presented a cobblestone shape.By immunofluorescence method,we detected both Ⅷ factor and CD31 antigens in HUVECs.Western blotting results showed that HUVECs expressed eNOS.Conclusion Protocol we′ve established is easy to acquire.we have also built a standard method to identify HUVEC u-sing morphology,marker molecule and key enzyme.%目的：建立一个详细、易于重复的人脐静脉内皮细胞原代培养操作流程。方法采用胶原酶消化法分离人脐静脉内皮细胞；用倒置显微镜观察细胞形态，免疫荧光法检测内皮细胞标志分子，Western blotting检测内皮细胞标志酶———一氧化氮合酶的含量。结果0．2％胶原酶Ⅰ在37℃条件下消化5～13 min可得到高纯度（95％）人脐静脉内皮细胞；在倒置显微镜下，人脐静脉内皮细胞呈典型的鹅卵石样，细胞核明显；Ⅷ因子和CD31的免疫荧光检测呈阳性；Western blotting结果显示，分离到的人脐静脉内皮细胞中有一氧化氮合酶持续合成。结论建立的人脐静脉内皮细胞原代培养和鉴定的操作流程简单易学，且在形态、标志分子和关键性标志酶3个方面提供细胞鉴定的方法和参考数据。