| Project/Area Number |
11470340
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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 |
KOHORK Kenjiro Nagoya City University, Medical School, Professor, 医学部, 教授 (30122047)
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| Co-Investigator(Kenkyū-buntansha) |
TOZAWA Keiichi Nagoya City University, Medical School, Assistant Professor, 医学部, 講師 (40264733)
HAYASHI Yutaro Nagoya City University, Medical School, Associate Professor, 医学部, 助教授 (40238134)
佐々木 昌一 名古屋市立大学, 医学部, 講師 (50225869)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥14,600,000 (Direct Cost: ¥14,600,000)
Fiscal Year 2001: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2000: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1999: ¥8,100,000 (Direct Cost: ¥8,100,000)
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| Keywords | urolithiasis / osteopontin / gene transfection / gene therapy / アンチセンス / 細胞接着 / リポフェクション |
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| Research Abstract |
Biochemical analyses of urinary stones have shown that 1 to 5 % of their weight consists of proteinaceous ingredients, and several studies have emphasized the importance of proteins in stone formation. We previously extracted the proteinaceous fraction from calcium oxalate and calcium phosphorus stones using EDTA and identified osteopontin (OPN) as one of the major constituents of urinary stones. In another study, we found a strong expression of OPN mRNA on distal tubular cells in the kidneys of stone-forming rats. OPN is clearly produced in the kidney, although its function and significance there remain unclear. As we speculated that OPN played a role in stone formation, we tried to block the production of OPN using transfection of antisense OPN to protect stone formation in the kidney. The Tetracycline-Regulated Expression System was used. Plasmid pTet-OPN as was constructed by cloning mouse OPN cDNA sequence in an inverted (antisense) orientation. Two clones of antisense transfectans were identified which showed marked reduction in OPN syntesis. 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 OPN RNA prevents the association of CaOx crystals with NRK-52E cells. Moreover, experimental kidney stone rat model in vivo transfection were inhibited OPN expression in the renal outer medulla where were expressed antisense OPN RNA, and were not observed the formation of CaOx microlith.
We suggest that the regulation of OPN protect the stone formation, and that OPN regulations is one of the methods of gene therapy to urolithiasis.
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