| Project/Area Number |
17591570
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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 | Kobe University |
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Principal Investigator |
NISHIDA Kotaro Kobe University, Graduate School of Medicine, Assistant Professor, 大学院・医学系研究科, 助手 (00379372)
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| Co-Investigator(Kenkyū-buntansha) |
DOITA Minoru Kobe University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (60237170)
AKISUE Toshihiro Kobe University, Graduate School of Medicine, Assistant Professor, 大学院・医学系研究科, 助手 (90379363)
KURODA Ryosuke Kobe University, University Hospital, Lecturer, 医学部附属病院, 講師 (80379362)
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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 |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2006: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | Intervertebral disc / RNA interference / gene therapy / Rat / in vivo / Fas ligand / 腰痛 / 組織再生 / 超音波 |
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| Research Abstract |
INTRODUCTION: RNA interference (RNAi) has recently emerged as an important biological strategy for gene silencing. The efficacy of RNAi mediated by Small Interfering RNAs (siRNAs) has been reported in nucleus pulposus cells in vitro using the exogenous reporter gene. We also reported the efficacy of micro-bubble enhanced ultrasound gene transfer (US gene transfer) to the disc in vivo. On the other hand, successful down regulation of endogenous gene by RNAi has not been reported. The objective of this study was 1) to demonstrate that specific gene expression can be suppressed in vivo by small interfering RNA (siRNA) mediated by US gene transfer, 2) to down regulate the expression of FasL, one of the genes reported to be endogenously expressed in NP cells. METHODS : 1) Two reporter luciferase plasmids (Firefly and Renilla) were used. These plasmids were co-transfected with siRNA targeting Firefly luciferase to the disc in vivo. The inhibitory effects were evaluated by dual luciferase assay for two weeks. 2) siRNA designed to specifically down regulate FasL were used. These siRNAs were transfected using the lipofection method. Seven days after transfection in vitro, cells were harvested and FasL expression was evaluated by western blotting. The house keeping gene (tubulin) expression was used as the internal control and non silencing siRNA was used for the negative control. The inhibitory ratio of the FasL expression against tubulin expression was calculated. RESULTS: 1) In the experimental group, the expression of Firefly luciferase was drastically inhibited in vivo. 2) At seven days, siRNA transfected group showed FasL expression was drastically down regulated in vitro compared with tubulin expression.
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