Application of in vitro Blood-Brain Barrier model for assessment of drug delivery to central nervous system.

(1)永生牛脑内皮细胞的建立为建立永生牛脑内皮细胞，将含有SV40T抗原的pSV3-neo导入原代培养的牛脑内皮细胞。经G418筛选，获得6个具有正常脑内皮细胞纺锤形形态的克隆。其中t-BBEC-117保留了脑内皮细胞的表型。(2)将永生大鼠星形胶质细胞pSV3-neo导入原代培养的大鼠星形胶质细胞。获得了4个表达胶质纤维酸性蛋白的不朽菌落。单个克隆ACT-57在特定的化学介质中生长稳定，NGF mRNA的表达强于正常星形胶质细胞。(3)星形胶质细胞诱导血脑屏障表型的鉴定。星形胶质细胞共培养可加强t-BBEC-117的细胞间接触。在已知的星形细胞因子中，只有碱性成纤维细胞生长因子(BFGF)能模拟星形胶质细胞的作用。这些数据表明，碱性成纤维细胞生长因子是诱导永生脑内皮细胞具有血脑屏障特性的最有可能的再生因子之一。

(1) Establishment of immortal bovine brain endothelial cellTo establish immortal brain endothelial cell, pSV3-neo which contains SV40 T antigen was introduced to primary cultured bovine brain endothelial cells. After selection by G418, we obtained six clones that exhibited the spindle-shaped appearance of normal brain endothelial cell. One of them, t-BBEC-117, retained the brain endothelial cell phenotype.(2) Establishment of immortal rat astrocytepSV3-neo was introduced to rat primary cultured astrocytes. We obtained four immortal colony, which exhibited glial fibrillary acidic protein. A single clone, ACT-57, exhibited stable growth in a chemically defined medium and a stronger expression of NGF mRNA than that of normal astrocyte.(3) Identification of astrocytic factor which induce blood-brain barrier phenotype.Astrocyte in co-culture was found to tighten the intercellular contacts of t-BBEC-117. Among known astrocytic factors, only fibroblast growth factor-basic (bFGF) could mimic the actions of astrocytes. These data suggest that bFGF is one of the most plausible astorcytic factors to induce the blood-brain barrier properties of immortal brain endothelial cells.