单筛过滤法体外分离培养原代大鼠脑微血管内皮细胞及其鉴定  

The primary culture and identification of rat brain microvascular endothelial cells isolated in vitro using single sieve filtration method

作　　者：张帆 李柏霖 刘海琴 唐元瑜[2] Zhang Fan;Li Boling;Liu Haiqin;Tang Yuanyu(College of Acupuncture and Moxibustion,Fujian University of TCM,Fuzhou 350122,China;College of TCM,Fujian University of TCM,Fuzhou 350122,China;Shanghai Long March Hospital Affiliated to Naval Medical University,Shanghai 200003,China)

机构地区：[1]福建中医药大学针灸学院,福州350122 [2]福建中医药大学中医学院,福州350122 [3]海军军医大学第二附属医院长征医院呼吸与危重症医学科,上海200003

出　　处：《中国组织化学与细胞化学杂志》2023年第4期402-406,共5页Chinese Journal of Histochemistry and Cytochemistry

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 采用单筛过滤法获取脑微血管以培养纯度高、活性好的原代大鼠脑微血管内皮细胞。方法 取4周龄SD大鼠脑皮质,经剪碎、单层细胞筛网过滤、收集网上截留组织、Ⅱ型胶原酶消化后,置于CO_(2)培养箱中进行原代培养。通过细胞形态学观察、第Ⅷ因子相关抗原免疫细胞化学染色检测鉴定所培养的目的细胞。结果 体外培养24 h后,短梭形细胞从脑微血管段周围爬出;48 h后“岛屿状”的细胞团簇形成;96 h后细胞融合,呈典型的单层、“铺路石样”、镶嵌式贴壁生长。第Ⅷ因子相关抗原免疫细胞化学染色检测显示细胞胞质呈现棕红色,表达为阳性。结论 单筛过滤法能够成功分离培养出原代大鼠脑微血管内皮细胞。Objective To obtain primary rat brain microvascular endothelial cells with high purity and good activity using a single sieve filtration method.Methods The cerebral cortex of 4-week-old SD rats was minced,filtered through a monolayer cell sieve,and the tissue retained on the net was collected and digested with type II collagenase,then placed in a CO2 incubator for primary culture.The target cells were identified by observing cell morphology and immunocytochemical staining for factor VIII-related antigen.Results Within 24 h of in vitro culture,short spindle-shaped cells migrated from around the segments of brain microvessels;after 48 h,island-like cell clusters formed;by 96 h,the cells fused,showing a typical monolayer,cobblestone-like,mosaic pattern of growth.Immunocytochemical staining for factor VIII-related antigen showed the cytoplasm in brown-red,indicating positive expression.Conclusion The single sieve filtration method can successfully isolate and culture primary rat brain microvascular endothelial cells.

关 键 词：脑 微血管内皮细胞 原代培养 单筛过滤法 Ⅷ因子相关抗原 

分 类 号：R-331[医药卫生]