A New therapeutic strategies for acute encephalitis by targeting astrocytes

Research Project

Project/Area Number	17K10121
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Multi-year Fund
Section	一般
Research Field	Pediatrics
Research Institution	Nagoya City University
Principal Investigator	Asai Kiyofumi 名古屋市立大学, 医薬学総合研究院(医学), 教授 (70212462)
Co-Investigator(Kenkyū-buntansha)	藤田政隆名古屋市立大学, 医薬学総合研究院(医学), 研究員 (10360637) 加藤晋名古屋市立大学, 医薬学総合研究院(医学), 助教 (90551250)
Project Period (FY)	2017-04-01 – 2021-03-31
Project Status	Completed (Fiscal Year 2020)
Budget Amount *help	¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000) Fiscal Year 2019: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000) Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000) Fiscal Year 2017: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Keywords	アクアポリン / 血液脳関門 / アストロサイト / 蛍光タンパク / 急性脳症 / 脳神経疾患 / サイトカイン
Outline of Final Research Achievements	To detect the cellular polarity of astrocyte, we employed aquaporin-4 (AQP4) expression as a marker of polarity. To detect the intracellular distribution of AQP4, pHluorin and mKate double-labeled AQP4 expression vector was constructed. pHluorin was introduced to the extracellular loop of AQP4 and mKate was fused to C-terminal. This vector was transfected to C6 astrocytoma and ACT57 immortalized rat astrocyte. Both green signal (pHluorin) and red signal (mKate) were detected 48 to 96 hrs after transfection. When the extracellular pH was changed from pH7.4 to pH6.0, the green signal was disappeared. This result might suggest AQP4 fusion protein was expressed on the plasma membrane. Next, the co-culture system of bovine brain endothelial cell line tBBEC-117 and ACT57 was tested. Clear cellular polarity was not detected at present.
Academic Significance and Societal Importance of the Research Achievements	本研究で構築できたAQP4と蛍光タンパクの融合タンパクを発現するベクターを用いることにより、アストロサイトの細胞極性の有無をリアルタイムに観察することが可能となると思われる。この方法を用いることにより、アストロサイトの活性化状態を観察することが可能となり、急性脳症の病態の解析やその治療法の開発に応用できるものと思われる。