REGULATIVE FACTORSOF WATER ABSORPTION IN HUMANENDOLYMPHATIC SAC

Research Project

Project/Area Number	11671686
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Otorhinolaryngology
Research Institution	NAGASAKI UNIVERSITY
Principal Investigator	SHIGENO Koichiro NAGASAKI UNIVERSITY, DEPARTMENT OF OTORHINOLARYNGOLOGY, ASSOCIATE PROFESSOR, 医学部, 助教授 (10162588)
Co-Investigator(Kenkyū-buntansha)	KOBAYASHI Toshimitsu NAGASAKI UNIVERSITY, DEPARTMENT OF OTORHINOLARYNGOLOGY, PROFESSOR, 医学部, 教授 (80133958) 中尾善亮長崎大学, 医学部・附属病院, 講師 (40188884)
Project Period (FY)	1999 – 2000
Project Status	Completed (Fiscal Year 2000)
Budget Amount *help	¥3,900,000 (Direct Cost: ¥3,900,000) Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000) Fiscal Year 1999: ¥3,000,000 (Direct Cost: ¥3,000,000)
Keywords	Endolymphatic Sac / Meniere's Disease / Endocytosis / Endolymphatic sac D.C.Potential / Gene Transfection / 遺伝子治療 / 培養
Research Abstract	1)An electronmicroscopic study was carried out on endolymphatic sac(ES) obtained from patients with Meniere's disease and autopsies. Severe degeneration was observed in many cases of Meniere's disease. However, it was speculated that ehdocytotic activities were still preserved in some cases. 2)Endolymphatic sac d.c.potential(ESP) was recorded in both patients with acoustic neuroma and with Meniere's disease. A verge value of the ESP in the patients with acoustic neuroma was 13.2±2.1mV.A patient with Meniere's disease shwed very low ESP value(2.4mV). The ES showed very severe degeneration in the patients with Meniere's disease. Our study could be the first successful measurement of human ESP. 3)Fluid phase endocytosis was investigated primary cultured endolymphatic sac of rat. K^+ depletion, K^+ concentration at 150mM, pH8.0, isoproterenol inhibited fluid phase endolymphatic sac. It is known that endolymph in the lumen of the ESP shows low K^+ concentration(15mM) and pH6.85. In such conditions, the cultured ES showed very active fluid phase endocytosis. 4)An transgenic mouse, the so-called Immorto-mouse(patent by the Charles River company) was used in order to make an inmortal cell line of the ES.This mouse has a temperature-sensitive copy of the large T-antigen of the SV40 virus(an oncogene) in its genome. The cells in an incubator at 33℃(permissive conditions) the T antigene is stable and the cells are conditional immortal. Then, when the cells are set to 39℃ the oncogene degrades and the cells differentiate. With the cell culture we could reproduced almost all of our PCR data published in Hearing Research(Beitz and Kumagami : expression pattern aquaporin water channels in the inner ear of the rat Hear Res 132, 76-84, 1999). 5)Gene transfection was carried out in an primary cultured endolymphatic sac of rat. cDNA of Green fluorescent protein(GFP) was transfected to the cultured endolymphatic sac