1998 Fiscal Year Final Research Report Summary
Basic research for gene therapy of urolithiasis by the inhibition of stone matrix protein
| Project/Area Number |
09470351
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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 |
Urology
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| Research Institution | Nagoya City University |
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Principal Investigator |
KOHRI Kenjiro Nagoya City University Medical School, chairman, professor, 医学部, 教授 (30122047)
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| Co-Investigator(Kenkyū-buntansha) |
SASAKI Shoichi Nagoya City University, assistant professor, 医学部, 講師 (50225869)
HAYASHI Yutaro Nagoya City University, assistant professor, 医学部, 講師 (40238134)
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| Project Period (FY) |
1997 – 1998
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| Keywords | urolithiasis / osteopontin / gene transfection / gene therapy / antisense |
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| Research Abstract |
Urinazy stones contain 1-5% protein, and many reports have suggested the importance of proteins in stone formation. We cloned and sequenced the cDNA encoding osteopontin (OPN), important soluble stone protein components of calcium oxalate stone proteins extracted with O.1M EDTA.Our previous study revealed that osteopontin (OPN), a secreted adhesive phosphoglycoprotein, were strongly expressed by renal distal tubular cells in the rat stone- forming kidney. OPN is clearly produced in the kidney, although its function and significance there remain unclear. Moreover, the association of crystals with renal tubular cells is considered a potential factor in the process of renal stone formation. The mechanisms, however, are not yet well understood. As we spaculated that OPN played a role in crystal-cell interaction, we tried to block the production of OPN by using normal rat kidney cell line (NRK-52E) transfected antisense OPN RNA.The Tetracycline-Regulated Expression System was used. Plasmid PTet-OPNas was constructed by cloning mouse OPN/2ar cDNA sequence in an inverted (antisense) orientation. Two clones of antisense transfectants were identified which showed marked reduction in OPN synthesis upon the absence of tetracycline. Intact NRK-52E cells, NRK-52E cells transfected with empty vector, and tetracycline-treated antisense clones, which did not show inhibition of OPN expression, all under identical conditions associated with calcium oxalate (CaOx) crystals. In contrast, the expression of antisense OPN12ar RNA prevents the association of CaOx crystals with >NRK-52E cells. We suggest that OPN plays a crucial role in this process.
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[Publications] Keiji Fujita, Kiyofumi Asai, Nobuyuki Honda, Takahiro Yasui, Yutaro Hayashi, Shoichi Sasaki, Seiiti Hirota, Shintaro Nomura, Tsuyoshi Soji, Taiji Katoh, Kenjiro Kohri: "Expression of antisense osteopontin RNA inhibits associatioon of calcium oxalate crystals with NRK-52E cells" J Biol Chem. (in press).
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