2002 Fiscal Year Final Research Report Summary
Identifying new aggrecanase and producing antibody and gene-targeting mouse
| 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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| Keywords | aggrecanase / extracellular matrix / rheumatoid arthritis / aggecan / matrix metalloproteinase |
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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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[Publications] Takemoto S, Murakami T, Kusachi S, Iwabu A, Hirohata S, Nakamura K, Sezaki S, Havashi J, Suezawa C, Ninomiya Y, Tsuji T.: "Increased expression of dermatopontin mRNA in the infarct zone of experimentally induced myocardial infarction in rats : comparison with decorin and type I collagen mRNAs"Basic Res Cardiol.. 97(6). 461-468 (2002)
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「研究成果報告書概要(欧文)」より
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[Publications] Komata T, Kondo Y, Kanzawa T, Ito H, Hirohata S, Koga S, Sumiyoshi H, Takakura M, Inoue M, Bama BP, Germano IM, Kyo S, Kondo S.: "Caspase-8 gene therapy using the human telomerase reverse transcriptase promoter for malignant glioma cells"Hum Gene Ther.. 13(9). 1015-1025 (2002)
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「研究成果報告書概要(欧文)」より
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[Publications] Komata T, Kondo Y, Kanzawa T, Hirohata S, Koga S, Sumiyoshi H, Srinivasula SM, Barna BP, Germano IM, Takakura M, Inoue M, Alnemri ES, Shay JW, Kyo S, Kondo S.: "Treatment of malignant glioma cells with the transfer of constitutively active caspase-6 using the human telomerase catalytic subunit (human telomerase reverse transcriptase) gene promoter"Cancer Res.. 61(15). 5796-5802 (2001)
Description
「研究成果報告書概要(欧文)」より
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