| Project/Area Number |
10557227
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
医薬分子機能学
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| Research Institution | TOHOKU UNIVERSITY |
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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)
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| 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
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| 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)
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| Keywords | blood-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 |
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| 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.
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