| Project/Area Number |
09671755
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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 |
Otorhinolaryngology
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| Research Institution | Kagoshima University |
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Principal Investigator |
MATSUNE Shoji Faculty of Medicine Kagoshima University, Assistant Professor, 医学部, 講師 (00253899)
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| Co-Investigator(Kenkyū-buntansha) |
HIRASE Hiroyuki University Hospital Kagoshima University, Research Associate, 医学部・附属病院, 助手 (30305130)
NISHIZONO Hirofumi Faculty of Medicine Kagoshima University, Research Associate, 医学部, 助手 (00284878)
KURONO Yuichi Faculty of Medicine Kagoshima University, Professor, 医学部, 教授 (80153427)
福田 勝則 鹿児島大学, 医学部・附属病院, 講師 (90156779)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1998: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1997: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | differential display method / cytokeratin / ciliogenesis / RPCS3a / H type 3 / floating culture / 繊毛細胞分化 / H type3 / 呼吸上皮細胞 / floating culture法 |
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| Research Abstract |
Genetic investigation : Several bands that appeared on the collagen gel following floating were extracted by the differential display method. The protein volume expression of 5 of the 25 sequenced bands was determined before and after floating by Northern blotting. Protein expression did not differ between the two measurement period. This lack of difference was attributed to the high frequency of false positive results using Northern blotting.
Morphological confirmation of cell differentiation : Expression of cytokeratin the cultured cells was examined chronologically by an immunohistochemical technique using anti-cytokeratin 8, 13 and 14 monochronal antibodies, although the cultured cells showed no positive findings before floating, this analysis showed a gradual increase in positive staining after floating in the following pattern ; anti-cytokeratin 8 was positively stained from the middle to apical layer of the cultured cells and anti-cytokeratin 13 was positively stained only in the middle layer and anti-cytokeratin 14 was positively stained in the basal layer.
Detection of ciliogenesis-specific substance : Several monochronal antibodies were produced, of which RPCS3a, the most eligible candidate specific to ciliogenesis, was characterized. RPCS3a was expressed in the cultured cell from the 3rd day after floating according to cellular ELISA.On the 10th day, RPCS3a was expressed most strongly, and many cultured cells contained secretory vesicles at their apical site as well as numerous centrioles indicating to be preciliated cells. ELISA revealed that RPCS3a is specific to the a sort of fucosylated carbohydrate H type 3. The expressed volume of H type 3 recognized by RPCS3a appears to reflect the stage of ciliogenesis. Based on the present data, we consider H type 3 to be a relativity specific markers of ciliogenesis and thus may provide a useful tool to clarify the mechanisms of ciliogenesis.
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