中文摘要：

目的建立简便、可靠、高效的体外分离培养人脐静脉内皮细胞（HUVEC）的方法。方法利用2型胶原蛋白酶消化处理脐带分离HUVEC,采用内皮细胞培养基（ECM）进行培养。相差倒置显微镜观察细胞形态,免疫荧光技术检测冯·维勒布兰德因子（v WF）的表达,流式细胞术检测细胞表面CD31的水平鉴定细胞的纯度,体外成管实验检测冻存后内皮细胞的功能。结果成功分离培养出纯度高、活性好的HUVEC。原代HUVEC分离1周即可汇合成单层,细胞呈典型的鹅卵石状排列,免疫荧光技术证明细胞广泛表达v WF、CD31阳性细胞频数达93.1%。冻存后的细胞依然能形成管腔结构,证明标准化的冻存方法能很好地维持细胞的功能。结论建立了简便、可靠、高效的体外分离培养HUVEC的方法。

英文摘要：

Objective To establish a simple,reliable and efficient isolation and culture method of human umbilical vein endothelial cells（ HUVECs） in vitro. Methods Type 2 collagenase was used to digest umbilical cord and separate HUVECs.The cells were cultured in the endothelial cell culture medium（ ECM）. The cell morphology was observed under an inverted phase-contrast microscope. Immunofluorescence technique was applied to detect the expression of von Willebrand factor（ v WF）. Cell purity was determined by detecting CD31 level on cell surface with flow cytometry. Tube formation assay was used to test the function of the endothelial cells after cryopreservation in vitro. Results HUVECs successfully isolated were proved with high purity and good activity. HUVECs of primary generation could merge into a single layer one week after isolation. The cells showed a typical cobblestone-like arrangement. Immunofluorescence technique validated that the cells could widely express v WF and the expression frequency of CD31 was 93. 1%. The cells were still capable of forming the lumen structure after cryopreservation,indicating that the standardized cryopreservation method could well maintain the cell function. Conclusion This is a simple,reliable and efficient method of isolating and culturing HUVECs in vitro.