小鼠脑微血管内皮细胞的原代培养与鉴定  

Primary culture and identification of mouse brain microvascular endothelial cells

作　　者：刘昭德 马华根 唐元瑜[2] LIU Zhaode;MA Huagen;TANG Yuanyu(College of Integrated Traditional Chinese and Western Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China;College of Traditional Chinese Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China)

机构地区：[1]福建中医药大学中西医结合学院,福建福州350122 [2]福建中医药大学中医学院,福建福州350122

出　　处：《细胞与分子免疫学杂志》2020年第4期325-329,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81072714);福建省自然科学基金(2017J01545)。

摘　　要：目的探索和建立一种纯度高、操作简单且重复性强的小鼠脑微血管内皮细胞原代培养方法。方法通过剪碎、过细胞筛网等物理方法,结合白蛋白密度梯度离心及化学酶消化法,并以严格控制1 g/LⅡ型胶原蛋白酶消化作用时间15~20 min为首要,从2~3周龄ICR小鼠脑组织中分离培养出小鼠脑微血管内皮细胞。通过细胞形态学观察和免疫细胞化学染色检测Ⅷ因子相关抗原鉴定所培养的细胞。结果接种24 h,贴壁的血管段周围爬出多角形或短梭状细胞,并逐渐扩散成团簇状;7 d后细胞融合,呈典型的单层、"铺路石样"镶嵌式排列。Ⅷ因子相关抗原细胞免疫化学染色鉴定为阳性,胞质呈棕红色,内皮细胞纯度达95%以上。结论成功建立了一种操作简单、重复性强、目的细胞纯度高的小鼠脑微血管内皮细胞原代培养方法。Objective To develop and establish a simple and repeatable method of primary culture of mouse brain microvascular endothelial cells(BMECs)of relatively high purity.Methods Isolated and cultured from the brain tissue of ICR mice aged 2-3 weeks old,the mouse BMECs were gained through physical fragmentation methods such as cutting up and passing through cell sieve,BSA density gradient centrifugation and chemical enzyme digestion.The digestion time of 1 g/L typeⅡcollagenase was strictly controlled within 15-20 minutes.The cultured cells were identified by cell morphological observation and immunocytochemical staining to detect factorⅧ-associated antigen.Results In 24 hours after seeding,the spindle-shaped or polygon-shaped cells began to migrate from the micro vessel segment and gradually gathered and grew in clusters;after 7 days,the fused cells showed a typical single layer and arranged in the shape of paving stone.Ⅷ-associated antigen showed positive in the cytoplasm which was brown red.The purity of microvascular endothelial cells was above 95%.Conclusion We have successfully established a simple and repeatable primary culture method for mouse BMECs of relatively high purity.

关 键 词：脑微血管内皮细胞 原代培养 Ⅷ因子相关抗原 免疫细胞化学染色法 

分 类 号：R392[医药卫生—免疫学] R392.12[医药卫生—基础医学]