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

应用体外血脑屏障模型评估中枢神经系统药物输送。

• 批准号: 11557107
• 负责人: ASAI Kiyofumi
• 金额: $ 8.19万
• 依托单位: Nagoya City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557107/
• 关键词: Blood-Brain Barrier; Endothelial Cell; Astrocyte; BBBモデル; 不死化; アストロサイト産生因子

(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.

(1)建立牛脑内皮永生细胞，将含有SV40 T抗原的pSV3-neo引入原代培养的牛脑内皮细胞中，建立牛脑内皮永生细胞。经过G418筛选，我们获得了6个具有正常脑内皮细胞梭形外观的克隆。其中的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的建立引入大鼠原代培养的星形胶质细胞。我们获得了4个具有胶质原纤维酸性蛋白的不朽菌落。单克隆细胞ACT-57在化学确定的培养基中表现出稳定的生长，并且比正常星形胶质细胞更强烈地表达NGF mRNA。(3)诱导血脑屏障表型的星形细胞因子的鉴定。共培养的星形胶质细胞使t-BBEC-117细胞间接触增强。在已知的星形胶质细胞因子中，只有碱性成纤维细胞生长因子（bFGF）能够模拟星形胶质细胞的作用。这些数据表明，bFGF是最可能诱导永生脑内皮细胞血脑屏障特性的星形胶质细胞因子之一。

项目成果

Morikawa, M., Asai, K., Kokubo, M., Fujita, K., Yoneda, N., Yamamoto, N., Inoue, Y., Iida, J., Kishimoto, T.and Kato, T.: "Isolation and characterization of a new immortal rat astrocyte with a high expression of NGF mRNA"Neurosci.Res.. 39. 205-212 (2001)

森川 M.、浅井 K.、小久保 M.、藤田 K.、米田 N.、山本 N.、井上 Y.、饭田 J.、岸本 T. 和加藤 T.：

Kataoka, H., Miura, Y., Joh, T., Seno, K., Tada, T., Tamaoki, T., Nakabayashi, H., Kawaguchi, M., Asai, K., Kato, T.and Ito, M.: "Alpha-fetoprotein producing gastric cancer lacks transcription factor ATBF1."Oncogene. 20. 869-873 (2001)

Kataoka, H.、Miura, Y.、Joh, T.、Seno, K.、Tada, T.、Tamaoki, T.、Nakabayashi, H.、Kawaguchi, M.、Asai, K.、Kato, T. 和

Tsuiki, H., Asai, K., Yamamoto, M., Fujita, K., Inoue, Y., Kawai, Y., Tada, T., Moriyama, A., Wada, Y.and Kato, T.: "Cloning of a rat glia maturation factor-gamma (rGMFG) cDNA and expression of its mRNA and protein in rat organs."J.Biochem.. 127. 517-523

Tsuiki, H.、Asai, K.、Yamamoto, M.、Fujita, K.、Inoue, Y.、Kawai, Y.、Tada, T.、Moriyama, A.、Wada, Y. 和 Kato, T.：

Sobue, K., Yamamoto, N., Yoneda, K., Fujita, K., Miura, Y., Asai, K., Tsuda, T., Katsuya, H.and Kato, T.: "Molecular cloning of two bovine a aquaporin-4 cDNA isoforms and their expression in brain endothelial cells."Biochim.Biophys.Acta. 1489. 393-398 (19

Sobue, K.、Yamamoto, N.、Yoneda, K.、Fujita, K.、Miura, Y.、Asai, K.、Tsuda, T.、Katsuya, H. 和 Kato, T.：“两个细胞的分子克隆

Yamamoto, M., Yoneda, N., Asai, K., Sobue, K., Tada, T., Fujita, Y., Katsuya, H., Fujita, M., Aihara, N., Mase, M., Yamada, K., Miura, Y.and T., K.: "Alterations in the expression of the AQP family in cultured rat astrocytes during hypoxia and reoxygenati

山本 M.、米田 N.、浅井 K.、Sobue, K.、多田 T.、藤田 Y.、胜也 H.、藤田 M.、相原 N.、马濑 M.、