| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Naoyuki Showa University, School of Dentistry, Assistant Professor, 歯学部, 講師 (90119222)
YAMAGUCHI Akira Showa University, School of Dentistry, Associate Professor, 歯学部, 助教授 (00142430)
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| Research Abstract |
The object of this Project is to investigate each function of the osteoclast and osteoblast during bone resorption process. We applied in vitro culture systems for studying the functions. The results obtained from this project are as follows.
(1)Function o-f the osteoblast a)Immunoreactive-collagenase was detected in all of the osteoblastic cell lines tested (5 clonal rat osteoblastic cell lines established by ourselves and a rat osteosarcoma cell line, UMR-106) using a rabbit anti-rat collagenase antibody by an indirect immunoperoxidase technique. b)Tissue plasminogen activator, which is a stimulator of latent form-collagenase, was also detected in all of the osteoblastic cell lines tested by an immunohistochemical technique. c) Collagenase activity was measured using as assay kit [collagenokit]. This kit contains fluorescence- conjugated collagen as substrate for collagenase. We found no significant increase of collagenase activity in the osteoblastic cell lines between basal level an
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d stimulated conditions with PTH or 1,25(OH)_2D_3. These results suggested to test the more sensitive assay system, including [14C] labeled collagen 'for the detection of the collagenase activity in the osteoblastic cells. d) We developed new assay system for the measurement of bone degradation;osteoblastic cells were cultured on the 13H]-proline labeled calvaria with or without 1,25(OH)_2D_3, and the level of 13H) was determined in the medium and bone after 5 days culture. As a result, 1,25(OH)_2D_3 stimulated the degradation of collagenous matrix by the osteoblastic cells.
a)Immunoreactive-collagenase was detected in all of the osteoblastic cell lines tested (5 clonal rat osteoblastic cell lines established by ourselves and a rat osteosarcoma cell line, UMR-106) using a rabbit anti-rat collagenase antibody by an indirect immunoperoxidase technique. b)Tissue plasminogen activator, which is a stimulator of latent form-collagenase, was also detected in all of the osteoblastic cell lines tested by an immunohistochemical technique. c) Collagenase activity was measured using as assay kit [collagenokit]. This kit contains fluorescence- conjugated collagen as substrate for collagenase. We found no significant increase of collagenase activity in the osteoblastic cell lines between basal level and stimulated conditions with PTH or 1,25(OH)_2D_3. These results suggested to test the more sensitive assay system, including [^<14>C] labeled collaben, for the detection of the collagenase activity in the osteoblastic cells. d) We developed new assay system for the measurement of bone degradation;osteoblastic cells were cultured on the [3H]-proline labeled calvaria with or without 1,25(OH)_2D_3, and the level of [3H] was determined in the medium and bone after 5 days culture. As a result, 1,25(OH)_2D_3 stimulated the degradation of collagenous matrix by the osteoblastic cells.
(2)Study for the osteoclast formation
a) We developed an in vitro assay system for the osteoclast formation from mouse bone marrow cells. b)Using this system, we found that 1,25(OH)_2D_3, PTH, IL-1, PGE_2 stimulate the osteoclast formation, and calcitonin and interferon-gamma inhibit its formation. c) We demonstrated that mouse spleen cells have a potential to develop the osteoclast. In this process, spleen cells required the presence of the osteoblastic cells, 1,25(OH)_2D_3 and dexamethasone. Less
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