天津医药

• 实验研究 • 上一篇    下一篇

人脐静脉血管内皮细胞的高效分离与培养

张臣1,李彤2,侯跃龙3,严晓晔3,朱争艳2,王鹏3   

  1. 1. 甘肃省人民医院心外科
    2. 天津市第三中心医院
    3.
  • 收稿日期:2009-10-16 修回日期:2010-03-15 出版日期:2010-07-15 发布日期:2010-07-15
  • 通讯作者: 李彤

Culture and identification of human umbilical vein endothelial cells in vitro

  • Received:2009-10-16 Revised:2010-03-15 Published:2010-07-15 Online:2010-07-15

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摘要： 摘要 目的：建立人脐静脉内皮细胞（HUVEC）体外分离、培养、鉴定的高效方法。方法：胰酶消化法从脐静脉获得HUVEC，观察其形态并传代培养，检测其摄取Dil标记的乙酰化低密度脂蛋白(Dil-AcLDL)的情况，传3代流式检测内皮细胞特异性表达。结果：消化分离的细胞呈小圆形且聚集成团；2 h细胞开始贴壁，呈条索状；7d细胞增多呈漩涡样生长；此后细胞继续增多接近融合，呈铺路石样改变。流式检测细胞高表达血管内皮细胞特异性标志CD144、vWF、CD31，部分表达CD34，而白细胞共同抗原CD45为阴性表达。镜下发现贴壁细胞（P1、P5）摄取Dil-AcLDL，胞浆呈现红色荧光，提示为有活性的内皮细胞。结论：利用胰酶消化法能从人脐静脉获得大量内皮细胞，体外培养后能成功增殖传代。

关键词: 内皮细胞, 脐静脉, 培养, 鉴定

Abstract: ABSTRACT Objective: To establish a effective method to isolate, culture and identify from human umbilical vein endothelial cells ( HUVEC) in vitro. Methods: Trypsin perfusion method was used to isolate HUVEC from fresh umbilical cord, and then the cells were cultivated in M199 with 20 % fetal bovine serum. After the collected endothelial cells were cultured and passaged, the cells were observed under inverted phase contrast microscope. The endothelial specific surface marker were assessed by fluorescence activated cell sorter ( FACS) analysis. And the cells were also identified by the test of up take of acety-lated low density lipoprotein (Ac-LDL). Results: The obtained HUVECs were small-round and clustering. The cells began adhere to the plate in tow hours, and attached cells were cord-like structure. After 7days culture the cells grow into large number and can be confluens a monolayer, which displayed cobble-stone morphology. These cells express endothelial specific markers, including VE-cadherin, vWF and CD31.The cells’function were identified by up take of Ac-LDL. Conclusion: A large number of high purified endothelial cells could be acquired by digestion of trypsin. HUVEC can be proliferated and passaged successfully in vitro.

Key words: endothelial cell, umbilical vein, culture, identification