プラズマ遺伝子導入の機序解明と作用要因のバランス制御による高効率非侵襲化

• Project Area: Plasma medical innovation
• Project/Area Number: 15H00896
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: Ehime University
• Principal Investigator: 神野 雅文 愛媛大学, 理工学研究科(工学系), 教授 (30274335)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥11,700,000 (Direct Cost: ¥9,000,000、Indirect Cost: ¥2,700,000); Fiscal Year 2016: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000); Fiscal Year 2015: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000)
• Keywords: プラズマ医療 / 遺伝子導入 / マイクロプラズマ / ラジカル / シナジー効果 / エンドサイトーシス / 遺伝子医療 / 育種 / プラズマ / プラズマ遺伝子導入法 / 遺伝子 / 形質転換 / マイクロキャピラリー

Outline of Annual Research Achievements

前年度に、プラズマ遺伝子導入の機序が分子量により異なることを示唆する実験結果を得たが、今年度は、実際にエンドサイトーシス阻害の有無による遺伝子導入率の変化を低分子のYO-YO1(～1.3kDa)と大分子のプラスミド(pCX-EGFP: 5,5kbp, 360万Da)で比較したところ、YOYO-1は変化がなく、プラスミドはエンドサイトーシス阻害により導入が80%低下して、通常の20%になったことから、YO-YO1などの低分子はエンドサイトーシス以外のプロセスで細胞に導入されており、プラスミドなどの大分子は主にエンドサイトーシスにより導入されていることが明確になった。
また、レーザ生成プラズマによりプラスミド溶液中に生成されるラジカルの定量をおこない、シナジー効果の検証をおこなった結果、プラズマ遺伝子導入には電気的要因と化学的要因の両方が不可欠であり、どちらか一方のみでは全く導入が生じないことが明らかになった。特に、電気的要因については電流が重要であることが示唆される結果が得られた。
これらのことから、電気的要因と化学的要因のシナジー効果によりプラズマ遺伝子導入がおこなわれていることが明確になった。この結果から、プラズマ遺伝子導入法は、細胞へのダメージを抑えて高効率で遺伝子を導入する条件を、標的細胞と導入物質に合わせてチューニングできるテイラーメイド的な汎用性を持つ手法であることが明らかになった。
標的細胞の多様化も進み、20種類以上の細胞に対してプラスミドの導入を確認している。また、国際共同研究を実施し、プラズマによるプラスミドの膜透過についてモンテカルロシミュレーションを用いた数値計算コードを開発した。

Research Progress Status

28年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

28年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] Universite de Toulouse 3, Paul Sabatier/Universite de Nantes(フランス)
• [Int'l Joint Research] ツールーズ大学(フランス)
• [Journal Article] Reactive oxygen species and intracellular Ca2+ contribution to micro-discharge plasma gene transfection (2017)
  • Author(s): Yuki Isozaki, Yoshihisa Ikeda, Yugo Kido, Susumu Satoh, Masafumi Jinno
  • Journal Title: Plasma Medicine Volume: -
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Synergistic effect of electrical and chemical factors on endocytosis in micro‐discharge plasma gene transfection (2017)
  • Author(s): Masafumi Jinno, Yoshihisa Ikeda, Hideki Motomura, Yuki Isozaki, Yugo Kido, Susumu Satoh
  • Journal Title: Plasma Sources Science and Technology Volume: -
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Optical emission spectroscopy characterizations of micro-air plasma used for simulation of cell membrane poration (2016)
  • Author(s): Amel Zerrouki, Hideki Motomura, Yoshihisa Ikeda, Masafumi Jinno, Mohammed Yousfi
  • Journal Title: Plasma Physics and Controlled Fusion Volume: 58 Issue: 7 Pages: 075006-075006
  • DOI: 10.1088/0741-3335/58/7/075006
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Monte Carlo poration model of cell membranes for application to plasma gene transfection (2016)
  • Author(s): Amel Zerrouki, Mohammed Yousfi, Ahmed Rhallabi, Hideki Motomura and Masafumi Jinno
  • Journal Title: Plasma Processes and Polymers Volume: 13 Issue: 6 Pages: 633-648
  • DOI: 10.1002/ppap.201500144
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Effects of molecular size and chemical factor on plasma gene transfection (2016)
  • Author(s): Yoshihisa Ikeda, Hideki Motomura, Yugo Kido, Susumu Satoh, Masafumi Jinno
  • Journal Title: Japanese Journal of Applied Physics Volume: 55 Issue: 7S2 Pages: 07LG06-07LG06
  • DOI: 10.7567/jjap.55.07lg06
  • NAID: 210000146890
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Improvement of efficiency and viability in plasma gene transfection by plasma minimization and optimization electrode configuration (2016)
  • Author(s): Masafumi Jinno, Kunihide Tachibana, Hideki Motomura, Noboru Saeki, Susumu Satoh
  • Journal Title: Japanese Journal of Applied Physics Volume: 55 Issue: 7S2 Pages: 07LG09-07LG09
  • DOI: 10.7567/jjap.55.07lg09
  • NAID: 210000146893
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Investigation of plasma induced electrical and chemical factors and their contribution processes to plasma gene transfection (2016)
  • Author(s): Masafumi Jinno, Yoshihisa Ikeda, Hideki Motomura, Yugo Kido, Susumu Satoh
  • Journal Title: Archives of Biochemistry and Biophysics Volume: 605 Pages: 59-66
  • DOI: 10.1016/j.abb.2016.04.013
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Improvement of efficiency and viability in plasma gene transfection by plasma minimization and optimizing electrodes configuration (2016)
  • Author(s): Masafumi Jinno, Kunihide Tachibana, Hideki Motomura, Noboru Saeki, Susumu Satoh
  • Journal Title: Japanese Journal of Applied Physiscs Volume: 55
  • NAID: 210000146893
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Effects of molecular size and chemical factor on plasma gene transfection (2016)
  • Author(s): Yoshihisa Ikeda, Hideki Motomura, Yugo Kido, Susumu Satoh, and Masafumi Jinno
  • Journal Title: Japanese Journal of Applied Physiscs Volume: 55
  • NAID: 210000146890
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] プラズマによる遺伝子導入 (2015)
  • Author(s): 神野雅文、佐藤晋
  • Journal Title: プラズマ・核融合学会誌 Volume: 91 Pages: 788-792
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] プラズマ照射によるスマ魚卵への蛍光色素導入 (2017)
  • Author(s): 大野 雄貴、船越 健太、木戸 祐吾、池田 善久、佐藤 晋、神野 雅文
  • Organizer: 第64回応用物理学会春季学術講演会
  • Place of Presentation: パシフィコ横浜, 横浜, 神奈川県
  • Year and Date: 2017-03-14
• [Presentation] プラズマによるC3H/HeJ マウス皮膚へのin vivo 遺伝子導入 (2017)
  • Author(s): 江渕 拓也、森 貴之、木戸 祐吾、池田 善久、佐藤 晋、神野 雅文
  • Organizer: 第64回応用物理学会春季学術講演会
  • Place of Presentation: パシフィコ横浜, 横浜, 神奈川県
  • Year and Date: 2017-03-14
• [Presentation] プラズマ照射による脂質二重膜の変形と分子透過過程の検討 (2017)
  • Author(s): 山本 健太、永岩 秀憲、木戸 祐吾、本村 英樹、佐藤 晋、神野 雅文
  • Organizer: 第64回応用物理学会春季学術講演会
  • Place of Presentation: パシフィコ横浜, 横浜, 神奈川県
  • Year and Date: 2017-03-14
• [Presentation] プラズマ照射によるタマネギ表皮細胞への蛍光色素導入 (2017)
  • Author(s): 西 雅明、大野 雄貴、木戸 祐吾、池田 善久、佐藤 晋、神野 雅文
  • Organizer: 第64回応用物理学会春季学術講演会
  • Place of Presentation: パシフィコ横浜, 横浜, 神奈川県
  • Year and Date: 2017-03-14
• [Presentation] プラズマ分子導入における活性酸素種の役割の導入分子量依存性 (2017)
  • Author(s): 池田 善久、磯崎 勇希、木戸 祐吾、佐藤 晋、神野 雅文
  • Organizer: 第64回応用物理学会春季学術講演会
  • Place of Presentation: パシフィコ横浜, 横浜, 神奈川県
  • Year and Date: 2017-03-14
• [Presentation] プラズマ遺伝子導入法 ～導入機序と必要とされるプラズマ特性の検討 (2016)
  • Author(s): 神野 雅文
  • Organizer: 「プラズマ科学における分光計測の高度化と原子分子過程研究の新展開」、「原子分子データ応用フォーラムセミナー」合同研究会
  • Place of Presentation: 核融合科学研究所, 土岐市, 岐阜県
  • Year and Date: 2016-12-20
  • Invited
• [Presentation] Investigation of discharge propagation on cell and plasmid suspension in plasma gene transfection (2016)
  • Author(s): Yugo Kido, Hideki Motomura, Yoshihisa Ikeda, Susumu Satoh, Masafumi Jinno
  • Organizer: AVS 63rd International Symposium & Exhibition
  • Place of Presentation: Nashville, TN
  • Year and Date: 2016-11-06
  • Int'l Joint Research
• [Presentation] The role of electrical and chemical factors in the molecular/gene transfection by micro-plasma irradiation (2016)
  • Author(s): Masafumi Jinno, Yoshihisa Ikeda, Hideki Motomura, Yugo Kido, Susumu Satoh
  • Organizer: AVS 63rd International Symposium & Exhibition
  • Place of Presentation: Nashville, TN
  • Year and Date: 2016-11-06
  • Int'l Joint Research / Invited
• [Presentation] The synergistic eect between electrical and chemical factors in plasma gene/molecule-transfection (2016)
  • Author(s): Masafumi Jinno
  • Organizer: 69th Annual Gaseous Electronics Conference
  • Place of Presentation: Bochum, Germany
  • Year and Date: 2016-10-10
  • Int'l Joint Research / Invited
• [Presentation] Membrane deformation and permeabilization caused by microplasma irradiation (2016)
  • Author(s): Hideki Motomura, Hidenori Nagaiwa, Kenta Yamamoto, Yugo Kido, Yoshihisa Ikeda, Susumu Satoh, Masafumi Jinno
  • Organizer: 69th Annual Gaseous Electronics Conference
  • Place of Presentation: Bochum, Germany
  • Year and Date: 2016-10-10
  • Int'l Joint Research
• [Presentation] プラズマ照射による脂質二重膜の変形と分子透過過程の検討 (2016)
  • Author(s): 永岩 秀憲、山本 健太、木戸 祐吾、池田 善久、本村 英樹、佐藤 晋、神野 雅文
  • Organizer: 第77回応用物理学会秋季学術講演会
  • Place of Presentation: 朱鷺メッセ, 新潟市, 新潟県
  • Year and Date: 2016-09-13
• [Presentation] プラズマ照射によるメダカ魚卵への蛍光色素導入 (2016)
  • Author(s): 池田 善久、大野 雄貴、西 雅明、船越 健太、木戸 祐吾、斎藤 大樹、後藤 理恵、松原 孝博、本村 英樹、佐藤 晋、神野 雅文
  • Organizer: 第77回応用物理学会秋季学術講演会
  • Place of Presentation: 朱鷺メッセ, 新潟市, 新潟県
  • Year and Date: 2016-09-13
• [Presentation] 複数の針電極放電によるプラズマ遺伝子導入法 (2016)
  • Author(s): 磯崎 勇希、大西 章仁、木戸 祐吾、池田 善久、本村 英樹、佐藤 晋、神野 雅文
  • Organizer: 第77回応用物理学会秋季学術講演会
  • Place of Presentation: 朱鷺メッセ, 新潟市, 新潟県
  • Year and Date: 2016-09-13
• [Presentation] 多種細胞におけるプラズマ遺伝子導入法の導入傾向 (2016)
  • Author(s): 中野 孝輝、宮本 展寛、木戸 祐吾、池田 善久、本村 英樹、佐藤 晋、神野 雅文
  • Organizer: 第77回応用物理学会秋季学術講演会
  • Place of Presentation: 朱鷺メッセ, 新潟市, 新潟県
  • Year and Date: 2016-09-13
• [Presentation] 大気圧非平衡プラズマのアプリケーションとしてのプラズマ遺伝子導入 (2016)
  • Author(s): 神野 雅文
  • Organizer: 化学工学会 第48回秋季大会
  • Place of Presentation: 徳島大学, 徳島市, 徳島県
  • Year and Date: 2016-09-06
  • Invited
• [Presentation] Synergistic effect between electrical and chemical factors in plasma gene transfection (2016)
  • Author(s): Masafumi Jinno, Yuki Isozaki, Ken Miyake, Masanori Kimura, Yugo Kido, Yoshihisa Ikeda, Hideki Motomura, Susumu Satoh
  • Organizer: 6th International Conference on Plasma Medicine
  • Place of Presentation: Bratislave, Slovakia
  • Year and Date: 2016-09-04
  • Int'l Joint Research
• [Presentation] Membrane permeabilization induced by microplasma irradiation (2016)
  • Author(s): Hideki Motomura, Hidenori Nagaiwa, Daijiro Aibara, Yugo Kido, Yoshihisa Ikeda, Susumu Satoh, Masafumi Jinno
  • Organizer: 6th International Conference on Plasma Medicine
  • Place of Presentation: Bratislave, Slovakia
  • Year and Date: 2016-09-04
  • Int'l Joint Research
• [Presentation] Cytotoxicity of plasma gene transfection (2016)
  • Author(s): Yoshihisa Ikeda, Yuki Isozaki, Yugo Kido, Hideki Motomura, Susumu Satoh, Masafumi Jinno
  • Organizer: 6th International Conference on Plasma Medicine
  • Place of Presentation: Bratislave, Slovakia
  • Year and Date: 2016-09-04
  • Int'l Joint Research
• [Presentation] Clathrin-mediated endocytosis contribution to plasma gene transfection (2016)
  • Author(s): Masafumi Jinno, Yuki Isozaki, Yugo Kido, Yoshihisa Ikeda, Hideki Motomura, Susumu Satoh
  • Organizer: 6th International Conference on Plasma Medicine
  • Place of Presentation: Bratislave, Slovakia
  • Year and Date: 2016-09-04
  • Int'l Joint Research
• [Presentation] High-efficient and high-survivability plasma gene transfection by minimizing plasma and optimizing electrodes configuration (2016)
  • Author(s): Hideki Motomura, Yoshihisa Ikeda, Kunihide Tachibana, Noboru Saeki, Susumu Satoh, Masafumi Jinno
  • Organizer: 6th International Conference on Plasma Medicine
  • Place of Presentation: Bratislave, Slovakia
  • Year and Date: 2016-09-04
  • Int'l Joint Research
• [Presentation] Evaluation of importance of H2O2 in gene transfection (2016)
  • Author(s): Yoshihisa Ikeda, Masanori Kimura, Yuki Isozaki, Yugo Kido, Hideki Motomura, Susumu Satoh, Masafumi Jinno
  • Organizer: 6th International Conference on Plasma Medicine
  • Place of Presentation: Bratislave, Slovakia
  • Year and Date: 2016-09-04
  • Int'l Joint Research
• [Presentation] Micro-air plasma characterizations from optical emission spectroscopy for gene transfection application (2016)
  • Author(s): Amel Zerrouki, Hideki Motomura, Yoshihisa Ikeda, Masafumi Jinno, Mohammed Yousfi
  • Organizer: 7th International Workshop on Plasma Spectroscopy
  • Place of Presentation: Meitetsu Inuyama Hotel, Inuyama, Japan
  • Year and Date: 2016-06-26
  • Int'l Joint Research
• [Presentation] Investigation of cell membrane permeation process in plasma molecule transfection using artificial cell (2016)
  • Author(s): Hideki Motomura, Daijiro Aibara, Hidenori Aibara, Yugo Kido, Yoshihisa Ikeda, Susumu Satoh, Masafumi Jinno
  • Organizer: The 33rd International Conference of Photopolymer Science and Technology
  • Place of Presentation: Makuhari Messe, Chiba, Japan
  • Year and Date: 2016-06-22
  • Int'l Joint Research
• [Presentation] プラズマ遺伝子導入; 何がすごいのか! (2016)
  • Author(s): 神野 雅文
  • Organizer: 第19回プラズマ技術研究会/第9回ミニマル3 DICファブ開発研究会
  • Place of Presentation: リファレンス駅東ビル, 福岡市, 福岡県
  • Invited
• [Presentation] The effect of the suspension cells in plasma gene transfection method (2015)
  • Author(s): Y. Isozaki, K. Nakano, Y. Ikeda, H. Motomura, Y. Kido, S. Satoh, K. Tachibana and M. Jinno
  • Organizer: 9th International Conference on Reactive Plasmas
  • Place of Presentation: Hawaii, USA
  • Year and Date: 2015-10-12
  • Int'l Joint Research
• [Presentation] Influence of the H2O2 in the plasma gene transfection method (2015)
  • Author(s): H. Nagaiwa, D. Aibara, Y. Ikeda, H. Motomura, Y. Kido, S. Satoh, K. Tachibana, M. Jinno
  • Organizer: 9th International Conference on Reactive Plasmas
  • Place of Presentation: Hawaii, USA
  • Year and Date: 2015-10-12
  • Int'l Joint Research
• [Presentation] Effect of the cell temperature in the plasma gene transfection (2015)
  • Author(s): Y. Ikeda, K. Manabe, Y. Isozaki, H. Motomura, Y. Kido, S. Satoh, K. Tachibana and M. Jinno
  • Organizer: 9th International Conference on Reactive Plasmas
  • Place of Presentation: Hawaii, USA
  • Year and Date: 2015-10-12
  • Int'l Joint Research
• [Presentation] Quantitative Investigation of Surface Charge of Liposome for Analysis of Plasma Gene Transfection Mechanism (2015)
  • Author(s): H. Motomura, Y. Ikeda, Y. Kido, K. Tachibana, S. Satoh and M. Jinno
  • Organizer: 10th Asian-European International Conference on Plasma Surface Engineering
  • Place of Presentation: Jeju, Korea
  • Year and Date: 2015-09-20
  • Int'l Joint Research
• [Presentation] Mechanism of Plasma Gene Transfection (2015)
  • Author(s): M. Jinno, K. Manabe, Y. Isozaki, Y. Ikeda, H. Motomura, Y. Kido, S. Satoh and K. Tachibana
  • Organizer: 10th Asian-European International Conference on Plasma Surface Engineering
  • Place of Presentation: Jeju, Korea
  • Year and Date: 2015-09-20
  • Int'l Joint Research
• [Presentation] Contribution of the Reactive Species to the Plasma Gene Transfection (2015)
  • Author(s): Y. Ikeda, H. Motomura, Y. Kido, S. Satoh, K. Tachibana and M. Jinno
  • Organizer: 10th Asian-European International Conference on Plasma Surface Engineering
  • Place of Presentation: Jeju, Korea
  • Year and Date: 2015-09-20
  • Int'l Joint Research
• [Presentation] Effect of the radical species for gene transfection by discharge plasma irradiation (2015)
  • Author(s): Y. Ikeda, H. Motomura, Y. Kido, S. Satoh, K. Tachibana and M. Jinno
  • Organizer: 22nd International Symposium on Plasma Chemistry
  • Place of Presentation: Antwerp, Belgium
  • Year and Date: 2015-07-05
  • Int'l Joint Research
• [Remarks] 電気エネルギー変換工学研究室（神野研究室）
  • URL: http://www.mayu.ee.ehime-u.ac.jp/