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Development of in vitro blood-brain barrier culture system from temperature sensitive SV40T antigen gene transgenic animal

Research Project

Project/Area Number 10557227
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field 医薬分子機能学
Research InstitutionTOHOKU UNIVERSITY

Principal Investigator

TERASAKI Tetsuya  New Industry Creation Hatchery Center, Tohoku University, Professor, 未来科学技術共同研究センター, 教授 (60155463)

Co-Investigator(Kenkyū-buntansha) KIKUCHI Akihiko  Institute of Biomedical Engineering, Tokyo Women's Medical College, Assistant Professor, 医学部, 助手 (40266820)
OBINATA Masuo  Institute of Development, Ageing and Cancer, Tohoku University, Professor, 加齢医学研究所, 教授 (10099971)
HOSOYA Ken-ichi  Graduate School of Pharmaceutical Sciences, Tohoku University, Assistant Professor, 大学院・薬学研究科, 助手 (70301033)
FUKUSHIMA Kiyomi  Pharmaceutical Research Laboratories, Taisho Pharmaceutical Co., Ltd., Researcher, 開発研究所・薬物動態研究室, 室長
Project Period (FY) 1998 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥13,600,000 (Direct Cost: ¥13,600,000)
Fiscal Year 1999: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1998: ¥9,500,000 (Direct Cost: ¥9,500,000)
Keywordsblood-brain barrier / brain capillary endothelial cell / temperature sensitive SV40T antigen gene / conditionally immortalized cell line / glucose transporter / tight junction protein / p53 protein / P-glycoprotein / トランスジェニック動物 / グルコース / 温度感受性SV40T抗原遺伝子導入トランスジェニック動物 / 脳 / 不死化細胞クローン / GLUT-1 / グルコース輸送
Research Abstract

The purpose of the present study is to develop the in vitro blood-brain barrier (BBB) system. A conditionally immortalized brain capillary endothelial cell line was established from the temperature sensitive SV40T antigen gene transgenic mouse and rats. Both cell lines had spindle fiber shape and expressed SV40T antigen gene product. The amount of SV40T antigen in the cell cultured at 33 degree was significantly greater than those of 37 and 39 degree. Growth rate at 33 degree was also greater than those of 37 and 39 degree. BBB selective marker, i.e., acetylated LDL receptor, alkalinephosphatase, gamma-glutamyltranspeptidase, was detected for the cell lines established. RT-PCR analysis revealed that these cell line express the mRNA of GLUT-1 and mdr1a. Western blot analysis also indicated the expression of GLUT-1 gene product and P-glycoprotein. Initial uptake rate of 3-O-methyl-D-glucose (3-OMG) by the mouse cell line was 2.6-5.3μl(min mg protein). The Michaelis constant was 6.6 mM which is very similar to the in vivo reported value, while the transport activity was approximately ten fold smaller than that of in vivo uptake rate at the BBB. Tight junction of the established cell lines was not strong enough for the transcellular transport study, whereas mRNA of tight junction proteins such as claudin-5, occludin, junctional adhesion molecule was detected by the RT-PCR analysis. In conclusion, we could establish conditionally immortalized cell line of brain capillary endothelial cell in mouse and rats. These cell lines exhibited at least a part of in vivo characteristics. The induction of tight junction protein gene products and transporter gene product would be a subject for the future projects.

Report

(3 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • Research Products

    (24 results)

All Other

All Publications (24 results)

  • [Publications] K.Kitazawa: "Efflux of taurocholic acid across the blood-brain barrier : Interaction with cyclic peptides"J. Pharmacol. Exp. Ther.. 286. 890-895 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] S.Song: "Modulation of the tumor disposition of vinca alkaloids by PSC 833 in vitro and in vivo"J. Pharmacol. Exp. Ther.. 287. 963-968 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T.Terasaki: "The blood-brain barrier efflux transporters as a setoxifying system for the brain"Adv. Drug Deliv. Rev.. 36. 195-209 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] S.Tanabe: "In vivo and in vitro evidence of blood-brain barrier transport of a novel catiionic arginine-vasopression fragment 4-9 analogue"J. Pharmacol. Exp. Ther.. 290. 561-568 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T.Kitazawa: "In Vivo and In Vitro Evidence of a Carrier-Mediated Efflux Transport System for Estrone-3-sulfate scross the Blood-Cerebrospinal Fluid Barrier"J. Pharm. Pharmacol.. (印刷中). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 高長ひとみ: "ヒトにおける薬物動態とその変動要因の基礎知識;薬物動態を決定する機能タンパク質、血液脳関門と血液脳脊髄液関門"月刊薬事臨時増刊号. (印刷中). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T.Terasaki: "Development of Brain Efflux Index(BEI)Method and Its Application to the Blood-Brain Barrier Efflux Transport Study. In"An Introduction to the Blood-Brain Barrier : Methodology and Biology""Ed.by W.M.Pardridge, Cambridge Univ. Press. 486 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T.Terasaki: "Tissue Engineering for Therapeutic Use 4"Ed.by Y.Ikada and Y.Shimizu, Elsevier Science B.V.(印刷中). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Kitazawa: "Efflux of taurocholic acid across the blood-brain barrier : Interaction with cyclic peptides"J. Pharmacol. Exp. Ther.. 286. 890-895 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] S. Song: "Modulation of the tumor disposition of vinca alkaloids by PSC 833 n vitro and in vivo"J. Pharmacol. Exp. Ther.. 287. 963-968 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Terasaki: "The blood-brain barrier efflux transporters as a detoxifying system for brain"Adv. Drug Deliv. Rev.. 36. 195-209 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] S. Tanabe: "In vivo and in vitro evidence of blood-brain barrier transport of a novel cationic arginine-vasopression fragment 4-9 analogue"J. Pharmacol. Exp. Ther.. 290. 561-568 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Kitazawa: "In vivo and in vitro evidence of a carrier-mediated efflux transport system for estorone-3-sulfate across the blood-cerebrospinal fluid Barrier"J. Pharm. Pharmacol.. (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 高長ひとみ: "ヒトにおける薬物動態とその変動要因の基礎知識;薬物動態を決定する機能タンパク質、e)血液脳関門と血液脳脊髄液関門"月刊薬事臨時増刊号. (印刷中). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Terasaki: "Development of brain efflux index (BEI) method and its application to the blood-brain barrier efflux transport study. In "an introduction to the blood-brain barrier ; methodology and biology""Ed. by W.M. Pardridge, Cambridge Univ. Press. 486. (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Terasaki: "Tissue Engineering for Therapeutic Use 4"Ed. by Y. Ikada and Y. Shimizu, Elsevier Science B.V.. (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] S.Tanabe: "In vivo and in vitro evidence of blood-brain barrier transport of a novel catiionic argininevasopression fragment 4-9 analogue"J. Pharmacol. Exp. Ther.. 290. 561-568 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] T.Kitazawa: "In Vivo and In Vitro Evidence of a Carrier-Mediated Efflux Transport System for Estrone-3-sulfate across the Blood-Cerebrospinal Fluid Barrier"J. Pharm. Pharmacol.. (印刷中). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] 高長ひとみ: "ヒトにおける薬物動態とその変動要因の基礎知識;薬物動態を決定する機能タンパク質、e)血液脳関門と血液脳脊髄液関門"月刊薬事臨時増刊号. (印刷中). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] T.Terasaki: "Tissue Engineering for Therapeutic Use 4"Y.Ikeda and Y.Shimizu,Elsevier Science B.V.(印刷中). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] T.Kitazawa: "Efflux of taurocholic acid across the blood-brain barrier:Interaction with cyclic peptides." J.Pharmacol.Exp.Ther.286. 890-895 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] T.Terasaki: "The blood-brain barrier efflux transporters as a detoxifying system for the brain." Adv.Drug Deliv.Rev.(in press). (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] S.Song: "Modulation of the tumor disposition of vinca alkaloids by PSC833 in vitro and in vivo." J.Pharmacol.Exp.Ther.287. 963-968 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] T.Terasaki: "Development of Brain Efflux Index(BEI)Method and Its Application to the Blood-Brain Barrier Efflux Transport Study:in“An Introduction to the Blood-Brain Barrier:Methodology and Biology"." W.M.Pardridge,Cambridge Univ.Press., 8 (1998)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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