| Project/Area Number |
11672257
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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 |
応用薬理学・医療系薬学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
TAKAKURA Yoshinobu Kyoto University, Graduate Sch.Pharm. Sci., Professor, 薬学研究科, 教授 (30171432)
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| Co-Investigator(Kenkyū-buntansha) |
YAMASHITA Fumiyoshi Kyoto University, Graduate Sch.Pharm. Sci., Associate Professor, 薬学研究科, 助教授 (30243041)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1999: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Keywords | gene therapy / DNA vaccination / plasmid DNA / macrophage / cellular uptake / scavenger receptor / CpG motif / tumor necrosis factor / インターロイキン-6 / ポリアニオン / 活性化 |
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| Research Abstract |
Plasmid DNA (pDNA) has become an important class of macromolecular agent suitable for non-viral gene therapy as well as DNA vaccination. In vivo application of pDNA is thought to be safer than that of viruses because there is less potential for adverse effects. However, concerns have been raised since there is increasing evidence suggesting that bacterial, but not mammalian, DNA activates immune competent cells, especially macrophages. However, the cellular uptake mechanism of pDNA by macrophages is not yet fully understood. In order to elucidate the mechanism, the binding and uptake of pDNA were studied in vitro using cultured Chinese hamster ovary cells expressing the class A scavenger receptor (SRA) and peritoneal macrophages from SRA-knockout mice. We found that pDNA binding and uptake in mouse peritoneal macrophages are mediated by a specific mechanism to some defined polyanions based on three-dimtensional structure of polyanions. Furthermore, we investigated the cytokine secretion induced by pDNA containing unmethylated CpG motifs complexed with cationic liposomes. A significant amount of tumor necrosis factor-α (TNF-α) was produced from the macrophages upon stimulation with the complex. However methylated pDNA and calf thymus DNA complexed with the cationic liposomes could induce TNF-α and IL-6 production, indicating that these responses were not dependent on CpG motifs. These results suggest that pDNA becomes active to stimulate the cultured macrophages in vitro through CpG motif independent manner when it is combined with the liposome formulations. These findings would be an important basis for optimization of pDNA delivery in gene therapy and DNA vaccination.
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