CAJ | 학술논문

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。
목적：응용원대배양적대서뇌미혈관내피세포（brain-microvessel endothelial cells，BMECs ）여뇌미혈관주세포（brain-microvessel pericytes，BMPC ）、성형효질세포（astro-cytes，AS）공배양건립가모의재체상태적체외혈뇌병장（blood-brain barrier，BBB）모형。방법원대분리、순화화배양대서BMECs、BMPC화AS，통과세포형태학화면역세포화학염색방법감정원대배양적세포，응용Millicell세포배양삽（공경0.4μm）건립5충불동류형적체외BBB모형，경과내피전조치（transendothelial electrical resistance，TEER）、형광소납통투성（sodium fluorescent，Na-FLU ）、감성린산매（AKP）화γ-곡안선전태매（γ-GT1）적표체측정이급양성약재체내화체외BBB통투량적상사성，비교평개기병장공능。결과원대배양적BMECs정전형적포로란석양결구，BMPC포체교대차정분지상，AS 유세장돌촉，포질교천；면역세포화학염색증실원대세포위목표세포；BMECs여BMPC、AS공배양후TEER치가체（478±25）Ω·cm2，Na-FLU 적표관삼투계수위[（8.23±0.78）×10－6]cm·s－1，AKP화γ-GT1표체분별위（6.90±0.27）금씨단위· g－1 Pro，（4.39±0.32）μg·g－1 Pro；양성약재체외BBB적표관삼투계수（apparent permeability coefficient，Papp ）여재체수거구유교호적상관성（R2＝0.92）。결론원대배양적대서BMECs여BMPC、AS공배양건립적체외BBB모형재형태、결구급병장공능방면구비BBB적기본특정，위연구BBB적생이학、병이학이급사선화합물제공료일충유용공구。
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.