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

以大鼠原代脑内皮细胞、星形胶质细胞和周细胞为材料，构建单培养、双培养和三共培养7种不同类型的血脑屏障模型。通过测量小分子量标记荧光素的跨内皮电阻和通透性来比较模型的屏障完整性。我们可以证实单培养的脑内皮单层不形成紧密的屏障。在共培养条件下，周细胞对荧光素的电阻比I型星形细胞高，通透性比I型星形细胞低。在三重共培养模型中，当内皮细胞和周细胞位于插入物多孔过滤膜的两侧，星形胶质细胞位于培养皿（BBB kit）的底部时，观察到最紧密的屏障。利用BBBkit，我们研究了在体外prion蛋白片段PrP106-126、amyloid-B肽（1-42）和缺氧（BBB kit疾病模型）刺激下，中枢作用药物对血脑屏障的影响和通透性。依达拉奉是一种临床广泛使用的抗脑水肿药物，可显著保护短暂性脑缺血引起的血脑屏障破坏。PPS是一种朊病毒疾病的候选药物，在PrP106-126下，PPS在血脑屏障上的渗透速度更快。西洛他唑增加缺氧和组胺作用下脑水肿体外模型血脑屏障试剂盒p -糖蛋白活性。因此，我们的血脑屏障检测试剂盒是病理条件下研究血脑屏障药物通透性的合适体外实验工具。

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.