摘要
目的介绍一种从大鼠脑组织中分离培养原代大鼠脑血管内皮细胞(BCEC)的方法。方法采用酶消化、滤网机械分离结合右旋糖苷密度离心的方法分离脑毛细血管段,接种在涂有明胶的培养皿培养BCEC。结果通过环境扫描电镜观察细胞形态,量子点超敏细胞免疫荧光鉴定,我们培养出来了纯度较高的原代BCEC。结论将分离的鼠脑毛细血管段置于涂有明胶的培养皿上培养原代BCEC的新方法比传统的培养法可以得到纯度更高的BCEC,为后期基于BCEC为载体的动物实验奠定了细胞基础。
Objective To introduce a method for primary culture and identification of brain capillary endothelial cells （BCECs）. Methods The cortical tissues （3-5 g） were isolated from the brain of SD rats within 5 days ＂after birth and digested with 0.25% trypsin at 37 ~G for 30 minutes. After dextran-40 density gradient centrifugation, the pellet was resuspended in Hank＇s balanced salt solution （HBSS）. Then the suspension was filtered through 150 p^m mesh and followed by 75p.m mesh. The capillary segments in the mesh were collected and then digested with lmg/ml type Ⅱ collagenase for 30 minutes. After centrifugation, the pellet was resuspended in DEEM plus 10% fetal bovine serum. The cells were then inoculated into 35 mm culture dish covered with gelatin and incubated at 37 ~C in 5% CO2 incubator. Results Five days after inoculation, the BCECs began to out-migrate from the capillary segments and to produce a clone of BCECs. A large number of microvilli covered over the BCECs could be found ＇after magnification of 3000 by scanning electron microscope. Positive expression of von Willebrand factor could be found in more than 90% of the cultured cells. Conclusions The primary cultured BCECs with high purity may be obtaned through inoculation of capillary segments isolated from the cerebral tissues of SD rats into culture dish covered with gelatin.
出处
《中国临床神经外科杂志》
2014年第1期37-39,共3页
Chinese Journal of Clinical Neurosurgery
基金
国家科学自然基金(30973077)
关键词
大鼠脑毛细血管内皮细胞
原代培养
鉴定
大鼠
Brain capillary endothelial cell
Primary culture
Identification
Rats
作者简介
通讯作者：赵洪洋，E-mail：hyzha0750@sina．com
