| Project/Area Number |
13680828
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Nerve anatomy/Neuropathology
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| Research Institution | Tokyo Women's Medical University |
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Principal Investigator |
SASAKI Hiroshi Tokyo Women's Medical University, School of Medicine, Professor and chairperson, 医学部, 教授 (10014177)
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| Co-Investigator(Kenkyū-buntansha) |
HONDA Yoshiko Tokyo Women's Medical University, School of Medicine, Instructor, 医学部, 助手 (40287313)
HAYAKAWA Toru Tokyo Women's Medical University, School of Medicine, Instructor, 医学部, 助手 (60322484)
SATO Fumi Tokyo Women's Medical University, School of Medicine, Associate Professor, 医学部, 教授 (60205961)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | Astrocye / Western blot / AFAP / Cell culture / Aquaporin / Kir4.1 / Chemiluminescence / PCR / ECL / aquaporin4 / プライマー設計 / Agarose電気泳動 / 大腸菌 / BLAST |
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| Research Abstract |
The aim of this study was to demonstrate the expression of aquaporin4 (AQP4), aquaporin5 (AQP5), aquaporin9 (AQP9) and Kir4.1 in rat primary astrocytes using Western blot.
Trypsinized new-born rat brain was harvested into culture flasks. After 3 to 4 passages, the cell was solubilized with sodium dodecyl sulfate (SDS) and dithiothreitol. The lysate was separated with SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was incubated with primary antibodies and the secondary antibody (Horseradish peroxidase labeled IgG), then generated chemiluminescence was detected with X-ray film.
Glial fibrillary acidic protein (GFAP) specific for astrocytes showed was strongly expressed at about 50KDa. According to the protein database SWISS-PROT, the molecular size of GFAP is 49.9KDa, and then we identified the band as GFAP. AQP4 showed dual band at about 30KDa and 60KDa. According to SWISS-PROT, AQP4 is produced two different molecular size of protein, because two initiation codons in AQP4 mRNA called M1 and M23, then produced protein were 34.5KDa and 32.5KDa.
AQP5 and AQP9 showed a very weak band at each 60KDa. According to SWISS-PROT, molecular size of AQP5 and AQP9 are 28.4KDa and 31.4KDa, respectively. As a result, this may be dimer of AQP5 and AQP9 in our experiments.
Kir4.1 showed a positive band at about 50KDa. SWISS-PROT showed that the molecular size of Kir4.1 was 42.4KDa. It was possible to detect Kir4.1 in cultured astrocytes because this discrepancy between our finding and SWISS-PROT data was very little.
The next plans are to make a quantitative analysis of aquaporin expression in cell fraction and a morphological analysis of localization of aquaporin in astrocytes under brain edema.
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