| Project/Area Number |
02454423
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional basic dentistry
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
SHIMOKAWA Hitoyata Tokyo Medical and Dental University, Department of Biochemistry, Associate Professor, 歯学部, 助教授 (80014257)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1991: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1990: ¥6,300,000 (Direct Cost: ¥6,300,000)
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| Keywords | Ameloblast / Odontoblast / Osteoblast / Calcification / Amelogenin / Enamelin / Collagenase / Root resorption / アメロジェニロン / フォスフォフォリン / オステオカルシン |
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| Research Abstract |
1. Analysis of amelogenin gene expression by in situ hybridization and immunohistochemistry.
To elucidate temporal and spatial patterns of amelogenin gene expression in the rat tooth germ, in situ hybridiazation and immunohistochemicalstudies were carried out. Wistar rats were perfused with 1% glutaraldehyde and the upper incisors with surrounding tissues were dissected. The tissues were decalcified and processed for paraffin sections. Amelogenin antisense RNA was synthesized by T7 polymerase from a 860 bp full-length cDNA template subcloned in pSPT18. [alpha^<35>S]UTP was added to the reaction mixture to yield probes with specific activities of 5x10^8 cpm/mug. The hybridization signals were detected over ameloblasts from presecretory to the begining of maturation stage. The cells of stratum intermedium and stellate reticulum did not show hybridization signals. In immunohistochemical staining of the sections, positive signals were observed in ameloblasts from presecretory t o late matur
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ation stage. These results indicate that ameloblasts from presecretory to the beginning of maturation stage express amelogenin gene but not in late maturation stage and ameloblasts of maturation stage resorb amelogenin molecules.
2. Regulation of collagenase gene expression in human osteosarcoma-derived osteoblastic cell lines.
The regulation of collagenase gene expression in the human osteosarcoma-derived osteoblastic cell lines MG-63, U2-OS and human fibroblasts cell line IMR-90 was investigated by Northern analysis. Exposure of quiescent MG-63, U2-OS and IMR-90 cells to TPA and 10% FCS resulted in the induction of mRNA encoding collagenase. EGF induced collagenase mRNA in the IMR-90 cell but not in the MG-63 and U2-OS cells. In the IMR-90 and MG-63 cells, EGF stimulated the transcription of the c-fos and c-jun genes encoding the transcriptional factors which interact directly with the promoter region of the human collagenase gene. PTH and 1, 25-dihydroxyvitamin D_3 did not increase the collagenase mRNA level in both osteosarcoma cells. IL-1beta induced collagenase and c-jun but not c-fos mRNA in the MG-63 cell.
3. Detection of collagenase mRNA in bovine root resorbing tissue by in situ hybridization.
It has been reported that active root resorbing tissue has a strong collagenolytic activity. in order to elucidate the type of collagenase-producing cells and the role of collagenase in root resorption, in situ hybridization of bovine root resorbing tissue sections were conducted with digoxigenin-labeled non-radioactive RNA probes. Collagenase mRNA expression was clearly observed in odontoclasts in addition to macrophages, fibroblasts, odontoblasts and cemento-blasts. Less
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