摘要
目的探讨获取大鼠脑微血管内皮细胞（BMEC）的简单、有效方法,为构建体外血肿瘤屏障（BTB）模型提供材料。方法采集出生3~5 d的Wistar胎鼠大脑皮质,应用酶消化法及葡聚糖离心法获得脑微血管段后,接种于培养皿中进行原代培养,采用倒置显微镜对所培养的细胞进行形态学观察;以Ⅷ因子相关抗原免疫组化染色法鉴定细胞;将BMEC与C6脑胶质瘤细胞共培养,构建体外BTB模型,并采用免疫组化法和免疫荧光法检测BMEC间紧密连接相关蛋白occludin的表达。结果体外培养2 h时脑微血管段贴壁,12-48 h见圆形生发中心形成,2-3 d单层内皮细胞自生发中心长出,4-5 d见较大单层内皮细胞团,5-7 d可见融合成片的内皮细胞单层,外观呈＂铺路石＂样;第Ⅷ因子免疫组化结果显示＂铺路石＂样细胞胞质呈棕黄色染色;紧密连接相关蛋白occludin的免疫组化和荧光结果证明共培养的BMEC间表达BTB的特性。结论本方法能成功地进行大鼠原代BMEC培养,构建大鼠体外BTB模型,进而应用于BTB的生理、生化及药理学研究。
Objective To explore a simple and repmducable method for the in vitro isolation and cultme of rat brain microvascular en- dothelial cells （ BMEC ）, so as to provide materials for the construction of in vitro blood tumor barrier （BTB） models. Methods Relatively pure cerebral microvessel fragments were obtained from the cortex of 3-5 days old wistar rats through dissection,enzyme digestion,and dextran centrifugation. Then, these fragments were seeded on dishes for prinmry culture. The morphology of BMEC was observed under an invert microscope. BMEC were identified by immunohistochemistry with factor VIII-associated antigen. In vitro BTB models were constructed by co- cultivation of BMEC with C6 glioma cells. Tile expression of fight junction-related protein occludin in BMEC of BTB was measured by im- munohistochemistry and immunofluorescenee. Results Segments of cerebral microvessels adhered to the dishes after 2 hours of in vitro cul- tivation,which consisted of round growth-centers after 12-48 hours of cultivation. Single cells then grew out from these growth-centers after 2- 3 days of cultivation. After another 4-5 days, clusters of cell monolayer appeared around the growth-centers. Ultimately, cells arranged as peb- ble-stone like in compact monolayer on day 5-7. Immunohistochemistry results showed positive staining of VIII factor antibody in the cyto- plasm of cultural cells. Immunohistochemistry and immunofluorescence staining of occludin confirmed that the BMEC co-cultured with C6 ghoma cells expressed the characteristics of BTB. Conclusion A relatively pure primary cultures of BMEC were established in this study. These BMEC could be used to develop in vitro BTB model systems for the physiology, biochemistry and pharmacology studies of BTB.
出处
《中国医科大学学报》
CAS
CSCD
北大核心
2012年第10期873-876,共4页
Journal of China Medical University
基金
国家自然科学基金资助项目(81172197
81171131
30973079)
高等学校博士学科点专项科研基金资助项目(20092104110015
20102104110009)
沈阳市科学技术计划项目计划(F10-205-1-37
F10-205-1-22)
关键词
大鼠
原代脑微血管内皮细胞
体外分离与培养
血肿瘤屏障
rat
prirrkary brain rnicmvascular endothelial cells
in vitro isolation and culture
blood tumor barrier
