摘要
目的寻找一种简单并稳定的原代小鼠脑微血管内皮细胞的培养方法。方法采用两次酶消化法及4℃梯度离心法获得细胞,光镜和电镜下观察细胞形态,用检测Ⅷ因子相关抗原等方法鉴定细胞。结果光镜下细胞呈多角形或短梭形,岛屿样聚集,随细胞生长及不断融合而出现"漩涡状"、"铺路石"样排列。电镜下观察可见紧密连接结构。Ⅷ因子相关抗原免疫荧光染色阳性。结论上述方法可获得纯度较高的小鼠脑微血管内皮细胞,为其原代培养提供了一种经济、简易、重复性好的操作方法,也为进一步的生理、生化、药理及体外血脑屏障或共培养等方面的研究提供了材料来源。
Objective To find a simple and steady method for primary culture of cerebral microvascular endothelial cells（CMECs）.Methods The cerebral cortices of 1-to 3-day newborn C57 mice were dissected and the meninges were removed carefully,followed by digestion with two kinds of enzymes and centrifugation at 4℃.The CMECs were harvested and observed under the inverted phase contrast microscopy and the transmission electron microscopy（TEM）.The growth curve was drawn through MTT assay and factor Ⅷ associated antigen was detected by using immunofluorescence analysis to characterize CMECs.Results After primary culture for 24 h,the cells appeared ＂polygon-shaped＂ or ＂short spindle-shaped＂ and clustered like islands.As the cells grew,they formed confluent monolayers demonstrating ＂swirl＂ or ＂cobblestone＂ morphology.The structure of tight junctions was observed under the TEM and the immunofluorescence staining revealed that the cells were positive for anti-factor Ⅷ.Conclusion Using the above method,a higher purity of CMECs was obtained.This economical,simple and repetitive primary culture could offer material to the further study of the properties in the aspects of physiology,biochemistry and pharmacology,as well as the function study of the in vitro model of blood-brain barrier or co-culture system.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2012年第6期731-734,共4页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
关键词
小鼠
脑微血管内皮细胞
原代培养
mouse
cerebral microvascular endothelial cell
primary culture
