| Project/Area Number |
12557127
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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 | 展開研究 |
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| Research Field |
Orthopaedic surgery
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| Research Institution | Kyoto Prefectural University of Medicine |
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Principal Investigator |
KUBO Toshikazu Kyoto Prefectural University of Medicine, the medical department, Professor, 医学部, 助教授 (20178031)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥12,600,000 (Direct Cost: ¥12,600,000)
Fiscal Year 2001: ¥5,300,000 (Direct Cost: ¥5,300,000)
Fiscal Year 2000: ¥7,300,000 (Direct Cost: ¥7,300,000)
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| Keywords | gene therapy / non-viral vector / synovitis / electroporation / ASK / in vivo / アデノウイルスベクター / apoptosis |
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| Research Abstract |
It is needed for us to explore safer vector for gene therapy in clinical use. So we chose non-virus vectors that are relatively safe. Firstly, LacZ gene was transduced to chondrocytes-like cell line (HCS2/8) by three types of cationic polymer and this gene expression was evaluated by X-gal staining and ONPG assay. Polyamidoamine (PAMAM) dendrimer was most effective in three, and it showed the usefulness of non-virus vector. Injections into rat joints with this cationic polymer and EBV-based plasmid vector were found to improve the transduction efficacy in vivo. Then we employed the efficacy of electroporation in vivo. EBV-based plasmid vector containing marker gene was injected into rat knee joints and different voltages of electric pulses were applied with a pair of special electrodes. The gene expression reached the highest level in 150V, and it was much better than with cationic polymers. We took an interest in apoptosis signal regulating kinase (ASK) -1 gene. The expression of this gene in cells was expected to induce apoptosis. Adenovirus vector containing ASK-1 gene was infected to the human rheumatoid synovious cells placed in monolayer culture. In Hoechst 33342 staining assay, 60 hours later, apoptosis was induced in about 50% of cells. In this study, we found the usefulness of the electroporation for gene transduction to synovious tissue in vivo, compared with other non-virus vectors and showed the possibility that ASK-1 would be effective for synovitis by transducing to synovium.
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