| Co-Investigator(Kenkyū-buntansha) |
KUBO Toshikazu Kyoto Prefectural University of Medicine, Medical Science, Professor, 医学研究科, 教授 (20178031)
MAZDA Osamu Kyoto Prefectural University of Medicine, Medical Science, Assistant Professor, 医学研究科, 助教授 (00271164)
INOUE Shigehiro Kyoto Prefectural University of Medicine, Medical Science, Assistant, 医学研究科, 講師 (10184748)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Hydrodynamics-based transfection is the method of gene transfer using naked-DNA, and is known in the efficiency in transgenesis. The purspose of this study is to examine the effects of hydrodynamics-based transfection to the joint disorder. First, we prepared two types of plasmid vectors containing luciferases gene (GL3). pGEG.GL3 is the Epstein-Barr virus(EBV)-based plasmid vector inducted nuclear antigen 1(EBNA1) gene and oriP alignment of EBV. Otherwise, pG.GL3 is the control plasmid which does not have EBNA1 gene and oriP. The Plasmid DNA dissolved in the phosphate buffered saline was injected intravenously from the tails of DBA-1 mice under the pressure. With the time courses after the induction, the mice were euthanized, and the liver and other organs were excised. Luciferases-assay was used to examine the gene expression and locarization in the excited organs. As a result, lusiferases gene was expressed remarkably in the liver, and the amount of gene expression was higher in the
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pGEG.GL3 group than control group. The term of gene expression was longer in the pGEG.GL3 group similarly. In the heart, lung, kidney, and spleen, the results were similar with the liver although the amount of lusiferases gene expression was lower than the liver. But the gene expression in the cartilage and synovium in the joints were very few. To promote the transduction efficiency of naked-DNA into the synovial membrane, we tried again the transgenesis with the use of electric pulsation. After injection of plasmid vector into the joints, several degrees of electric pulsation were applied to the joints with the electrode on the skin. As a result, expression of transgene was observed specifically at the site of electric pulsation. Moreover, the highest gene expression was achieved at 150V of electric pulsation.
Collagen-induced arthritis(CIA) mouse is prepared by injecting bovine type-II collagen intravenously with the Freund complete adjuvant from the tail of DBA-1 mouse. CIA mouse is generally used as a model of rheumatoid arthritis(RA). Otherwise, it is well known that Th1-immune response has some roles in the pathogenesis of RA, and the interleukin(IL)-10 has the suppressive effect to the Th1-immune response. Using hydrodynamics-based transfection method, we examined the effects of transduction of IL-10 gene to the CIA mice. As a result, the onset of arthritis has been inhibited in the curative treatment groups, in which transduction of IL-10 gene was applied at 3, 4, and 5 weeks after the immunization by type-II collagen. From this result, we considered that the onset of arthritis has been inhibited by the suppressive effect of IL-10 to the Th1-immune response.
Hydrodynamics-based transfection method does not need either virus or compound macromolecules as a carrier. Moreover, the procedure is convenient and the cost for operation is cheap. Hydrodynamics-based transfection should be a innovative method for the gene therapy to the joint disorder. Less
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