| Project/Area Number |
13557217
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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 |
応用薬理学・医療系薬学
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
HARASHIMA Hideyoshi Hokkaido Univ., Grad. School of Pharmaceutical Sci., Prof., 大学院・薬学研究科, 教授 (00183567)
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| Co-Investigator(Kenkyū-buntansha) |
MIZUGUCHI Hiroyuki National Institute of Health Sciences, Researcher, 研究員 (50311387)
KAMIYA Hiroyuki Hokkaido Univ., Grad. School of Pharmceutical Sci., Asso. Prof., 大学院・薬学研究科, 助教授 (10204629)
MATSUDA Akira Hokkaido Univ., Grad. School of Pharmaceutical Sci., Prof., 大学院・薬学研究科, 教授 (90157313)
SUZUKI Yousuke Hisamitsu Pharmaceutical Co., Inc., Researcher, 研究員
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥15,600,000 (Direct Cost: ¥15,600,000)
Fiscal Year 2003: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2002: ¥4,700,000 (Direct Cost: ¥4,700,000)
Fiscal Year 2001: ¥6,600,000 (Direct Cost: ¥6,600,000)
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| Keywords | intracellular trafficking / endocytosis / adeno-virus / transcriptional efficiency / quantification / 共焦点レーザー顕微鏡 / エンドソーム / ライソゾーム / 非ウイルスベクター / ウイルスベクター / 細胞内動態制御 / 体内動態制御 |
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| Research Abstract |
Since endosomal escape and the nuclear delivery of plasmid DNA constitute major barriers to trans-gene expression, a quantitative evaluation of intracellular trafficking of the plasmid DNA would be highly desirable in terms of optimizing a non-viral gene delivery system. In the present study, a novel strategyis proposed for quantifying the intracellular localization of rhodamin-labeled plasmid DNA in endosomes/lysosomes, cytosol and the nucleus. Endosomes/lysosomes and nucleus were stained with LysoSensor DND-189 and Hoechst 33258, respectively, to distinguish them from the cytosol. Using this approach, the intracellular trafficking of plasmid DNA was analyzed after transfection with LipofectAMINE PLUS, steaiylated octaarginine (STR-RS) and R8. In the case of R8, most of the plasmid DNA was trapped by endosomes/lysosomes. STR-R8 enhanced endosomal escape and following nucleartranslocation time dependently. LipofectAMINE PLUS was much effective in rapidly delivering DNA to the nucleus as well as the cytosol, even 1 hr after transfection. These differences in the intracellular trafficking of plasmid DNA correlated well with the transfection activities of these non-viral systems. Therefore, this method has the potential of quantitatively analyzing the intracellular pharmacokinetics of plasmid DNA after transfection by non-viral systems and promises to provide useful information for optimizing non-viral gene delivery systems.
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