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

用原代大鼠脑内皮细胞、星形胶质细胞和周细胞在培养板上构建了7种不同类型的血脑屏障模型，即单培养、双培养和三培养。通过测量跨内皮电阻和小分子量标记物荧光素的渗透性来比较模型的屏障完整性。我们可以证实，在单一培养的脑内皮细胞单层不形成紧密的屏障。在共培养条件下，周细胞比I型星形胶质细胞诱导更高的电阻和更低的荧光素渗透性。在三重共培养模型中，当内皮细胞和周细胞位于插入物的多孔滤膜的两侧并且星形胶质细胞位于培养皿的底部（BBB试剂盒）时，观察到最紧密的屏障。我们使用BBB试剂盒，在体外朊病毒蛋白片段PrP 106 -126、淀粉样蛋白B肽（1-42）和缺氧刺激下（BBB试剂盒疾病模型），研究了中枢作用药物对BBB的影响和渗透性。依达拉奉是一种临床广泛使用的抗脑水肿药物，可以显着防御由于短暂性脑缺血而导致的BBB破坏。PPS是朊病毒疾病的候选药物，在PrP 106 -126下更快地穿透BBB。西洛他唑增加缺氧和组胺下BBB试剂盒的P-糖蛋白的活性，这是脑水肿的体外模型。因此，我们的血脑屏障试剂盒被认为是一个合适的体外实验工具，研究药物渗透性的血脑屏障在病理条件下。

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.