| Project/Area Number |
16591519
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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 | Louis Pasteur Center for Medical Research |
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Principal Investigator |
KISHIDA Tsunao Louis Pasteur Center for Medical Research, Research Center of Medical, PhD., Researcher, 分子免疫研究部, 研究員 (00370205)
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| Co-Investigator(Kenkyū-buntansha) |
KISHI Atsuko Louis Pasteur Center for Medical Research, Research Center of Medical, PhD., Researcher, 基礎研究部門, 研究員 (80260165)
KOKUBO Aoi Louis Pasteur Center for Medical Research, Research Center of Medical, Researcher, 基礎研究部門, 研究員 (60342705)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2004: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | atrophy of skeletal muscle / Cytokine / Molecular therapy / Myostatin / Regenerative Medicine |
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| Research Abstract |
Myostatin is secreted from muscle cells and blocks differentiation and proliferation of myoblasts, as a regulatory mechanism to maintain the number and volume of muscle fibers. We hypothesized that artificial inhibition of myostatin may induce regeneration and hypertrophy of skeletal muscle, providing novel therapeutic procedures to facilitate recovery of motility function in bedridden elderly whose muscles have been attenuated by long-term disuse. In this study we assessed feasibility of this molecular therapeutic strategy by administering short interfering RNA (siRNA) targeting myostatin into murine skeletal muscle. Synthetic siRNA duplex was labeled with Cy3 and mixed with RNAiFect (Qiagen) at various ratios. The resultant complex was transfected into cultured cells and fluorescent microscopic observation was performed to determine the optimal transfection condition as well as efficiency of delivery. As the results, more than 90% of the cells were successfully transfected with the s
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iRNA when 0.5 microgram of siRNA in combination with 3 microlitter of RNAiFect was added to ten thousand cells. Several siRNA duplexes were synthesized corresponding to various portions of the myostatin cDNA sequence (siMyostatin) and the siRNAs as well as a nonspecific siRNA as control were transfected into cultured cells under the optimal condition. Myostatin mRNA levels were estimated by Real-Time-PCR analysis to determine the most effective siRNA sequence for silencing of the target gene. The most remarkable silencing effect was obtained with siRNA that caused approximately 88% reduction in myostatin expression, while another siRNA, siMyostatin-2 was also comparably effective. Next, the siMyostatin-1 was transfected into C2C12 myoblast cell line and cell proliferation rate was estimated. Compared with untreated cells, or the cells given nonspecific siRNA, the siMyostatin-transfected cells showed significantly more vigorous proliferation. Based on these findings from the in vitro experiments, we examined transcriptional silencing effect in vivo of RNAi on myostatin expression. The siMyostatin-1 was labeled with Cy3 and transfected into the anterior tibial muscle of mice by means of electroporation. Fluorescence microscopic observation revealed that 60 to 90% of muscle cells contained the nucleic acid. Myostatin transcript was estimated by Real-Time PCR analysis, after transfection of siMyostatin-1 by the same protocol, and it was found that the message level of the protein was reduced to approximately 40% compared with that in the muscle electro-transfected with control siRNA. These results collectively indicate that specific knockdown of myostatin can be effectively achieved using the present methodologies, which may be applicable to RNAi-based molecular targeting therapy to promote muscular hypertrophy in bedridden elderly. Less
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