体外血脑屏障模型的建立及功能测定  

The establishment and function characteristics of blood brain barrier model in vitro

作　　者：陈雪[1] 王军[1] 王伟[1] 骆翔[1] 喻志源[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院神经内科,武汉430030

出　　处：《卒中与神经疾病》2014年第4期195-198,202,共5页Stroke and Nervous Diseases

基　　金：国家自然科学基金项目(81000521;81030021)

摘　　要：目的利用大鼠原代微血管内皮细胞及星形胶质细胞建立体外血脑屏障模型,并通过跨内皮细胞电阻(Trans-epithelium electrical resistant,TEER)方法对血脑屏障模型进行功能测定。方法原代分离纯化SD大鼠脑微血管内皮细胞和星形胶质细胞,用免疫荧光检测内皮细胞标志物VWF,紧密连接蛋白ZO-1,星形胶质细胞标志物GFAP;用微血管内皮细胞和星形胶质细胞在Transwell小室上建立体外血脑屏障模型,观察TEER值的动态变化。结果原代的微血管内皮细胞培养至融合后具有典型的梭形"铺路石"样外观,VWF鉴定细胞纯度达到95%以上,ZO-1免疫荧光鉴定证实细胞间形成紧密连接;原代培养的星形胶质细胞呈现具有多个突起的典型形态,GFAP鉴定细胞纯度达到95%以上;在第10 d,单独微血管内皮细胞血脑屏障模型的TEER值为(42±1.41)Ωcm2,内皮细胞和星形胶质细胞共培养血脑屏障模型的TEER值为(65±1.42)Ωcm2。结论建立了体外血脑屏障模型,通过TEER值测定证明共培养使模型更加完整,更加接近在体血脑屏障模型的特性。Objective To establish in vitro blood brain barrier model using microvascular endothelia cells and astrocytes of primary rat,and identify the model by measuring TEER value.Methods Primary SD rat microvascular endothelial cells and astrocytes were isolated and purified.The endothelial cell marker VWF,tight junction proteins ZO 1,the astrocyte marker GFAP were detected using immunofluorescence techniques.An in vitro blood brain barrier model was established in Transwell inserts using endothelial cells and astrocytes coculture system.We observed the dynamic change of TEER,studied the establishment and characteristics of the blood brain barrier.Results Once fusing,primary microvascular endothelial cells presented a typical fusi form cobblestone appearance.More than 95％ of endothelial cells were VWF positive.ZO 1 confirmed the form of tight junctions between endothelial cells.Primary astrocytes presented a typical morphology with many pocesses.More than 95％ of astrocyes were GFAP positive.The TEER of microvascular endothelial cell alone model was （42 ± 1.41） Ωcm2,while the TEER of the coculture model was （65 ± 1.42） Ωcm2.Conclusions Vitro blood brain barrier model was established.By the TEER value measurement we prove that coculture system of the blood brain barrier model was more complete,more close to the characteristics of the blood brain barrier in vivo.

关 键 词：血脑屏障 微血管内皮细胞 星形胶质细胞 跨内皮细胞电阻 

分 类 号：R741[医药卫生—神经病学与精神病学]