中文摘要：

目的：应用原代培养的大鼠脑微血管内皮细胞（brain-microvessel endothelial cells，BMECs ）与脑微血管周细胞（brain-microvessel pericytes，BMPC ）、星形胶质细胞（astro-cytes，AS）共培养建立可模拟在体状态的体外血脑屏障（blood-brain barrier，BBB）模型。方法原代分离、纯化和培养大鼠BMECs、BMPC和AS，通过细胞形态学和免疫细胞化学染色方法鉴定原代培养的细胞，应用Millicell细胞培养插（孔径0.4μm）建立5种不同类型的体外BBB模型，经跨内皮电阻值（transendothelial electrical resistance，TEER）、荧光素钠通透性（sodium fluorescent，Na-FLU ）、碱性磷酸酶（AKP）和γ-谷氨酰转肽酶（γ-GT1）的表达测定以及阳性药在体内和体外BBB通透量的相似性，比较评价其屏障功能。结果原代培养的BMECs呈典型的铺路卵石样结构，BMPC胞体较大且呈分枝状，AS 有细长突触，胞质较浅；免疫细胞化学染色证实原代细胞为目标细胞；BMECs与BMPC、AS共培养后TEER值可达（478±25）Ω·cm2，Na-FLU 的表观渗透系数为[（8.23±0.78）×10－6]cm·s－1，AKP和γ-GT1表达分别为（6.90±0.27）金氏单位· g－1 Pro，（4.39±0.32）μg·g－1 Pro；阳性药在体外BBB的表观渗透系数（apparent permeability coefficient，Papp ）与在体数据具有较好的相关性（R2＝0.92）。结论原代培养的大鼠BMECs与BMPC、AS共培养建立的体外BBB模型在形态、结构及屏障功能方面具备BBB的基本特征，为研究BBB的生理学、病理学以及筛选化合物提供了一种有用工具。

英文摘要：

Aim To establish in vitro blood-brain barrier （BBB） model with characteristics of simulation of in vivo BBB by primi-tive co-culture of brain-microvessel endothelial cells （BMECs） with brain-microvessel pericytes （BMPC）and astrocytes （AS）. Methods BMECs,BMPC and AS from SD rats were primitively isolated,purified and cultured,and then primitive culture cells were identified by cellular morphological and immunocytochemi-cal staining methods.Five types of in vitro BBB models were es-tablished by using Millicell culture insert （pore diameter 0.4μm）and their barrier functions were evaluated by detection of transendothelial electrical resistance （TEER）,permeability of sodium fluorescent （Na-FLU ）,expression of alkaline phospha-tase （AKP）and γ-glutamyl transpeptidase （γ-GT1 ）,and simi-larity of permeation amount for positive drugs in vitro and in vivo BBB conditions.Results Primitive culture of BMECs presented typical pebbles-like structure,BMPC presented larger soma with branching property,AS presented slender synapse and shallower cytoplasm.Moreover,immunocytochemical staining results iden-tified primitive cells were targeted cells.TEER value for co-cul-ture of BMECs,BMPC and AS reached （478 ±25 ）Ω· cm2 , permeability coefficients （Papp ）value of Na-FLU was [（8.23 ± 0.78） ×10 -6 ]cm·s-1 ,expression of AKP and γ-GT1 were （6.90 ±0.27 ）King unit · g-1 Pro and （4.39 ±0.32 ）μg · g-1 Pro respectively.Moreover,good correlation could be found in Papp for positive controls in vitro and in vivo BBB models （R2=0.92）.Conclusion The established in vitro BBB model by using primitive co-culture of BMECs with BMPC and AS posses-ses in vivo BBB properties in cell morphology,structures and barrier functions,and can be used as a powerful tool for studying physiology,pathology of BBB and screening candidate com-pounds.