| Project/Area Number |
17591589
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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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 | Osaka City University |
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Principal Investigator |
TERAI Hidetomi Osaka City University, Graduate School of Medicine, Lecturer, 大学院医学研究科, 講師 (20382046)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAOKA Kunio Osaka City University, Graduate School of Medicine, Professor, 大学院医学研究科, 教授 (30112048)
KAZUKI Keniti Osaka City University, Graduate School of Medicine, Associate Professor, 大学院医学研究科, 助教授 (80254407)
HASHIMOTO Yusuke Osaka City University, Graduate School of Medicine, senior resident, 大学院医学研究科, 研究医 (10382178)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2005: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | BMP / Noggin / siRNA / Electroporation / 再生医学 / 遺伝子 |
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| Research Abstract |
Noggin is one of major extracellular antagonists of bone morphogenetic proteins(BMPs). It is reported that noggin is upregulated in undifferentiated mesenchymal cells and myoblasts in response to BMPs, and this may cause diminish the performance of BMPs in bone formation. In this study, we investigated the effect of noggin silencing by short interfering RNA in osteogenic differentiation in vitro, and the ectopic bone formation in vivo. Noggin expression induced by BMP-4 in C2C12 cells, a myoblastic cell line, was comfirmed by real-time RT-PCR. Noggin mRNA expression was elevated by BMP-4 stimulation in dose-and time-dependent manner. We designed double-stranded short interfering RNA(siRNA) for targeting noggin. Plasmid vectors expressing noggin and siRNA were co-transfected into C2C12 cells, and successful specific suppression of noggin protein expression was confirmed by Western blot. Transfection of the noggin siRNA into C2C12 cell also suppressed the endogenous noggin mRNA expressio
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n induced by BMP-4, and enhanced BMP-4 induced alkaline phosphatase activity up to two fold. In vivo effect of noggin siRNA gene transfer on rhBMP-2 induced bone formation was examined by ectopic bone formation assay in mice. Noggin siRNA plasmid was injected into the dorsal muscle and electropolation procedures was applied to the muscle, and then collagen disk containing of rhBMP-2 was implanted into the muscle. On days 4 after surgery, total RNA was extracted from muscle tissue around the disk for real time RT-PCR analysis. Electroporation-mediated transfer of noggin siRNA markedly decreased expression of noggin mRNA, which increased after implantation of the collagen disk containing rhBMP-2. At 3 weeks after surgery, the implants were harvested and radiographed with a soft X-ray apparatus, and bone mineral content(BMC) of each ossicle was measured. The size of new bone induced by rhBMP-2 and the mean BMC were significantly increased by noggin siRNA gene transfer. Conclusively, these findings suggest that noggin siRNA gene transfer enhances the osteoblast differentiation induced by BMP-4 in vitro and the bone formation induced by BMP-2 in vivo. While further study is needed, this approach may be useful tool in clinical application. Less
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