| Co-Investigator(Kenkyū-buntansha) |
FUJIMURA Kazunobu Univ. of Occupational and Environmental Health, School of medicine, Assistant Professor, 医学部, 講師 (90299628)
UDAKA Tsuyoshi Univ. of Occupational and Environmental Health, School of medicine, Instructor, 医学部, 助手 (10369069)
HASHIDA Koichi Univ. of Occupational and Environmental Health, School of medicine, Instructor, 医学部, 助手 (90389461)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2003: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
1) Analysis of nasal polyp contents by SDS-polyacrylamide gel electrophoresis
Nasal polyps were surgically excised from patients with chronic sinusitis, washed with saline, and dissected to collect content effusion. The effusion was diluted with phosphate buffered saline, and centrifuged to remove cellular components. The supernatant was subjected to 12.5% SDS-polyacrylamide gel electrophoresis to analyze protein composition. Protein components did not enter to the separating gel from the stacking gel, indicating a high content of macromolecules such as glycosaminoglycans. The supernatant was then treated with hyaluronidase, chondroitinase or keratinase, and again subjected to SDS-polyacrylamide gel electrophoresis. However, the same result was obtained, implying low contents of hyaluronic acid, chondroitin sulfates and keratan sulfate.
2) Exocytosis of secretary vesicles of nasal polyp epithelial cells
Surgically excised nasal polyps were double-stained with a cell membrane marker, FM4-6
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4, and a secretory vesicle marker, lysotracker green, and was observed under a confocal laser scanning microscope. The excitation wavelength was 488 nm, and the emission wavelengths were 650 nm (FM4-64) and 540 nm (lysotracker green). Green fluorescence was gradually replaced by red fluorescence, suggesting the occurrence of endocytosis-exocytosis cycle. Observation of a vertical plane showed no evident exocytosis on the basolateral surface of the epithelial cells.
3) Measurement of short circuit current across the epithelial layer of the nasal polyp by Ussing chamber method
Nasal polyps were placed in the Ussing chamber, and short circuit current was measured to estimate an active ion transport. Some polyps showed a short circuit current of about 20 μA/cm^2 with a resistance of about 100 Ωcm^2, but most showed no detectable short circuit current with a resistance of 15-40 Ω cm^2. This suggest that an active ion transport may exist across the nasal polyp epithelial layer, but the short circuit current may be undetectable because of the impairment of the tight junction between the epithelial cells.
4) Observation of the tight junction and the leakage of a tracer at the ultrastructural level.
Transmission electron microscopy revealed an irregularity of the tight junction of the epithelial cells. Biotinylated phospholipid was loaded on the apical surface of the nasal polyp followed by the addition of avidin-gold. The sample was then examined under a transmission electron microscope, however, no obvious leakage was seen in the intercellular space between the epithelial cells. Less
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