| Project/Area Number |
13470312
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Orthopaedic surgery
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| Research Institution | OKAYAMA UNIVERSITY |
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Principal Investigator |
HIROHATA Satoshi Okayama University Graduate School of Medicine and Dentistry, Assistant, 大学院・医歯学総合研究科, 助手 (90332791)
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| Co-Investigator(Kenkyū-buntansha) |
YONEZAWA Tomoko Okayama University Graduate School of Medicine and Dentistry, Assistant, 大学院・医歯学総合研究科, 助手 (30304299)
OOHASHI Toshitaka Okayama University Graduate School of Medicine and Dentistry, Lecturer, 大学院・医歯学総合研究科, 講師 (50194262)
NINOMIYA Yoshufumi Okayama University Graduate School of Medicine and Dentistry, Professor, 大学院・医歯学総合研究科, 教授 (70126241)
MOMOTA Ryusuke Okayama University Graduate School of Medicine and Dentistry, Assistant, 大学院・医歯学総合研究科, 助手 (80263557)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥14,000,000 (Direct Cost: ¥14,000,000)
Fiscal Year 2002: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2001: ¥9,900,000 (Direct Cost: ¥9,900,000)
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| Keywords | aggrecanase / extracellular matrix / rheumatoid arthritis / aggecan / matrix metalloproteinase / ADAMTS / メタロプロテアーゼ |
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| Research Abstract |
To investigate new aggrecanase, we have done the following experiments and got some data.
1) Various ADAMTS-specific primers were designed and RT-PCR was performed. We cultured human chondrosarcoma cell lines OUMS-27 (Okayama University Medical School-27) and stimulated with interleukine-1 beta (IL-1β). To determine gene expression quantitatively, we employed real-time RT-PCR method. Briefly, total RNA was extracted and then DNAse treatmeat was done to eliminate contaminating genomic DNA. After reverse transcribed with random primers and enzymes, cDNA was served as a template for RT-PCR. GAPDH was used for the internal control.
2) The expression and gene regulation by IL-1β was different amonf the ADAMTS-1,4, and -5, which were reported to have aggrecan cleaving property in vitro. We also investigated other ADAMTS gene expressions.
3) We identified another up-regulating ADAMTS gene by IL-1β in OUMS-27 cells. We raised polyclonal antibody against this new ADAMTS gene using peptide sequence of this ADAMTS.
4) We also started to making knock-out mouse for this gene. We screened mouse genomic library and identified several clones including this new ADAMTS gene. Under the collaboration with Dr. Apte's lab in the USA, we started to put our clones to ES cells.
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