| Project/Area Number |
16591508
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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 |
KAZUKI Kenichi (2005) Osaka City University, Graduate School of Medicine, assistant professor, 大学院・医学研究科, 助教授 (80254407)
長山 隆一 (2004) 大阪市立大学, 大学院・医学研究科, 助手 (50336781)
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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)
香月 憲一 大阪市立大学, 大学院・医学研究科, 助教授 (80254407)
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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 |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2004: ¥3,200,000 (Direct Cost: ¥3,200,000)
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| Keywords | poly-D, L-lactic acid and polyethylene glycol (PLA-PEG) / Bone Morphogenetic Protein / Gene therapy |
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| Research Abstract |
We studied the effectiveness of gene transfer using the biodegradable polymer (PLA-PEG). Beta-galactosidase and luciferase were used to assess the efficiency of gene transfer into muscle in vivo. LacZ gene was inserted under the chick actin promoter on pCAGGS expression vector which were highly expressed the inserted gene in muscle cells. Luciferase was expressed using pGL3-control vector^<【○!R】> which was including SV40 promoter. In our previously studies, we demonstrated that cationic lipid lipofection, Lipofectamine 2000^<【○!R】> (LF2000) was effective to transfer the plasmids into cultured mesenchymal stem cells. In order to increase the gene transfer efficiency by PLA-PEG in vivo, we mixed LF2000 with PLA-PEG. To determine the effectiveness of PLA-PEG with or without LF2000 to transfer plasmids into muscle, the mice were divided into three groups based on pellets received, i.e., (1)45mg of PLA-PEG containing 15μl of LF2000 and 45μg of plasmids ; (2)45mg of PLA-PEG containing 45μg of plasmids ; and (3)45mg of PLA-PEG only. Under general anesthesia, the PLA-PEG containing plasmid was implanted into the left dorsal muscle pouches of mice. One week after implantation, the mice were sacrificed and the tissues around the implanted pellets were harvested. The expression level of β-galactosidase in pellets was histologically evaluated by X-gal and hematoxylin-eosin stains. In addition, luciferase activity in extracted specimens from pellets was measured.
X-gal stain in LF2000+ plasmids + PLA-PEG pellets was stronger than that in plasmids + PLA-PEG. In the histological study, the area of X-gal staining was located in the cells around the pellet but not in muscle cells. Positive cells with X-gal staining were similar with mesenchymal stem cells. The expression of luciferase in the LF2000+ DNA+ PLA-PEG pellet was higher than that in DNA+ PLA-PEG.
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