A new blood-brain barrier(BBB) model : Specific uses under in vitro conditions of brain diseases

以原代培养的大鼠脑内皮细胞、星形胶质细胞和周细胞为材料，构建了7种不同类型的血脑屏障(BBB)模型，即单一培养、双重和三重共培养。通过测量小分子标志物荧光素的跨内皮电阻和通透性来比较模型的屏障完整性。我们可以证实，脑内皮细胞单层在单一培养中不会形成紧密的屏障。在共培养条件下，周细胞比I型星形胶质细胞对荧光素有更高的电阻和更低的通透性。在三重共培养模型中，当内皮细胞和周细胞位于贴片的多孔滤膜两侧，星形胶质细胞位于培养皿(BBB试剂盒)的底部时，屏障最紧密。使用BBKIT，我们研究了在PrP106-126、淀粉样蛋白B(1-42)和低氧(BBB KIT疾病模型)的体外刺激下，中枢作用药物对BBB的影响和通透性。临床上广泛使用的抗脑水肿药物依达拉奉显著地防御了短暂性脑缺血引起的BBB中断。在PrP106-126的作用下，PrP106-126下的PPS在血脑屏障上的渗透速度更快。西洛他唑提高低氧和组胺条件下血脑屏障试剂盒的P-糖蛋白活性，组胺是一种体外脑水肿模型。因此，我们的血脑屏障试剂盒是研究病理条件下血脑屏障药物通透性的一种合适的体外实验工具。

Seven different types of blood-brain barrier(BBB)models, mono-culture, double and triple co-cultures, were constructed from primary rat brain endothelial cells, astrocytes and pericytes on culture inserts. The barrier integrity of the models were compared by measurement of transendothelial electrical resistance and permeability for the small molecular weight marker fluorescein. We could confirm that brain endothelial monolayers in mono-culture do not form tight barrier. Pericytes induced higher electrical resistance and lower permeability for fluorescein than type I astrocytes in co-culture conditions. In triple co-culture models the tightest barrier was observed when endothelial cells and pericytes were positioned on the two sides of the porous filter membrane of the inserts and astrocytes at the bottom of the culture dish(BBB kit). With the BBBkit, we investigated effects and permeability on the BBB of centrally acting drugs under in vitro stimulations of prion protein fragment PrP106-126, amyloid-B peptide(1-42), and hypoxia(BBB kit disease models).Edaravone, a clinically-widely used anti-brain edema drug, markedly defended the BBB disruption due to a transient cerebral ischemia. PPS, a candidate drug for prion disease, penetrated more rapidly on the BBB under PrP106-126. Cilostazol increased the activity of P-glycoprotein of the BBB kit under hypoxia and histamine, an in vitro model of the brain edema. Thus, our BBB kit was found to be a suitable in vitro-experimental tool to study drug-permeability on the BBB under pathological conditions.