| 研究課題/領域番号 |
25253004
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| 研究機関 | 京都大学 |
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研究代表者 |
HEUSER John 京都大学, 物質-細胞統合システム拠点, 教授 (40571815)
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| 研究分担者 |
諸根 信弘 京都大学, 物質-細胞統合システム拠点, 講師 (50399680)
村上 達也 京都大学, 物質-細胞統合システム拠点, 准教授 (90410737)
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| 研究期間 (年度) |
2013-05-31 – 2016-03-31
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| キーワード | Electron microscopy / drug delivery / endocytosis / transfection |
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| 研究実績の概要 |
The basic goal of this project has been to visualize by electron microscopy (EM), the basic mechanism of entry of the important cell-penetrating therapeutic agents, so that we can better understand their mechanism of action, and thereby develop improved therapeutics for the future. We initially started with the general hypothesis that everyone in the field is following: that the entry of such therapeutics is via endosome rupture, after they have entered cells by endocytosis.
However, despite our greatest efforts, we have failed over the past two years to obtain any drug-delivery materials, which could be good enough to enter cells and be seen by EM nor have we managed to produce such materials by ourselves.
Therefore, we had no choice but to go all the way back to the beginning, and determine exactly how the 'classical' DNA transfection-reagents work, the process that labs all over the world depend upon every day. We started by visualizing in the EM how the most widely used transfection reagent, Lipofectamine, interacts with cells. We discovered that it is not endocytosed effectively but that it adheres to the cell surface in a very nasty way that sorely damages the cell membrane.
This has given us a fascinating glimpse into fundamental mechanisms of membrane healing and has pointed out the huge problems with using transfection-agents like Lipofectamine. Now, we are in a position to rationalize the development of new, much less harmful transfection methods, which should ultimately be very useful for medical research and medical therapeutics.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Cell-penetrating therapeutic agents obtained from different laboratories failed to work for our EM, and our own development of uptake-complexes proved to be too difficult, so we chose to study the standard transfection-reagent used worldwide, Lipofectamine, which no one has ever looked by EM.
Immediately, we obtained very impressive EM-views of Lipofectamine-DNA complexes on the surfaces of tissue-cultured cells, which we promptly named “injectosomes," since it was quite apparent from our EM's that these assemblies remain on the cell surface for long periods.
Most importantly, we observed in the EM clear signs that Lipofectamine 'injectosomes' on cell surfaces must be damaging the cell membrane repeatedly and incessantly, probably due to their massive size, their tight apposition to the membrane, and their spidery appearance.
Additionally, looking at longer times after exposure to Lipofectamine, we detected other remarkable structural-changes in the cells: changes that included most especially a gross over-abundance of caveolae on the surfaces of cells and distinctly abnormal deployments of microvilli and other actin-rich structures on Lipofectamine-transfected cells.
All these EM observations not only help to clarify the fundamental mechanism by which Lipofectamine mediates DNA-entry into cells, which has heretofore been entirely mysterious, but more importantly, they point toward ways that we can hope to improve transfection-efficiency and minimize cell damage during transfection.
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| 今後の研究の推進方策 |
This year (FY 2015) we plan to study more deeply the mechanism of action of all the other commercially available cationic liposome/DNA complexes that people use to transfect cells, as well as Lipofectamine. To this end, we have started collaborations with several of the people who originally invented these agents, and they are all very eager to help us understand what is going on.
This we will continue to do by our unique form of EM which we call "deep-etching", as well as by the more 'classical' approach of embedding cells in plastic and thin-sectioning, to learn more about the terrible oxidative damage that is going on inside of them during and after the membrane-damage provoked by transfection. This will allow us to better characterize what is going on with 'injectosomes' after they are endocytosed, and whether it is they or their degradation-products that are causing all the changes in plasma membrane cholesterol and caveolae, as well as all the long-term changes in the membrane cytoskeleton. Thereby, we hope to determine whether all these changes are ultimately due to some underlying mechanism of oxidative damage to the cells.
Finally, we will focus especially on developing new methods of transfection that lessen the damaging effects of these 'classical' reagents, in order to assist this field in the short-term, while we continue to search for wholly new transfection-reagents that do not exert so much membrane damage, in the first place.
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