摘要
目的:探索一种简便易行、可培养出高纯度大鼠脑微血管内皮细胞的方法。方法:1月龄SD大鼠,解剖得到大脑皮质,密度离心法获得较纯的脑微血管段后进行原代培养,传代采用差速消化和贴壁方法进行纯化。通过形态学观察及第Ⅷ因子相关抗原免疫荧光检测对培养细胞进行鉴定。结果:密度离心法分离出的大量微血管段呈"串珠样"结构,培养24h可见短梭形、多角形细胞,8～10天基本融合。第2代细胞经免疫荧光染色,第Ⅷ因子相关抗原呈阳性,阳性率达90%。结论:原代细胞采用低分子量葡聚糖加Percoll密度离心法、传代细胞采用差速消化和差速贴壁法纯化可成功培养高纯度的脑微血管内皮细胞。
Objective:This study was to establish a method to culture brain microvascular endothelial cells(BMECs). Method:Isolating BMECs from neonatal Sprague Dawley rats by density gradient centrifugation using low molecular weight dextran and 45% Percoll. Then we acquired a great deal of microvessels which like the 'string-of-beads' and seeded them in culture plate. The BMECs were pured by velocity digest and sedimentation in sub-cultured. The cell morphology was observed under phase-contrast microscope and demonstrated by immunofluorescence for VIII factor protein.Results:The characterization of BMECs was confirmed based on the morphology and immunofluorescence technology.More than 90% of cultured cells were positive for VIII factor protein,which indicated the cultured cells were vascular endothelial cells.Conclusion:In our study,the rat BMECs were successfully cultured by density gradient centrifugation using low molecular weight dextran and 45% Percoll in primary,velocity digest and sedimentation in sub-cultured.
出处
《微循环学杂志》
2011年第1期3-5,F0002,I0001,共5页
Chinese Journal of Microcirculation
基金
国家自然科学基金(NO.30770573)
北京市自然科学基金(NO.7092013)
北京市教育委员会科技发展计划项目(KM200710025007)
北京农学院兽医学(中医药)北京市重点实验室
2010年度开放课题(BUA-TCVM-201002)
关键词
脑微血管
内皮细胞
细胞培养
大鼠
Brain microvessel
Endothelial cell
Cell culture
Rat
