岡田 健吾¹, 工藤 信樹¹, 丹羽 光一², 山本 克之¹

Kengo OKADA¹, Nobuki KUDO¹, Koichi NIWA², Katsuyuki YAMAMOTO¹

¹北海道大学大学院情報科学研究科生体システム工学講座, ²北海道大学電子科学研究所

¹Graduate School of Information Science and Technology, Hokkaido University, ²Research Institute for Electronic Science, Hokkaido University

キーワード : sonoporation, pulsed ultrasound, microbubbles, cell membrane damage, cell membrane repair

はじめに：Sonoporationとは, 細胞に超音波を照射することにより細胞膜の透過性を一時的に向上させ, 通常では入らない薬物を細胞内へ導入する技術を言う. 最近の研究では微小気泡を付加するとパルス超音波でもsonoporationが生じることが報告されているが, 発生機序や基本的な性質は不明な点が多い. 本研究ではその機序と発生頻度および細胞膜損傷の修復について検討した結果について述べる. 方法：顕微鏡観察下で微小気泡と培養血管内皮細胞の位置関係を明確にした上でパルス超音波を照射し, 微小気泡が細胞におよぼす作用を高速度カメラを用いて観察した. 次に, 細胞膜損傷および修復の発生頻度を蛍光観察法により評価した. 結果：機序に関して, パルス超音波照射により生じる細胞近傍の微小気泡の膨張・収縮が細胞に直接機械的作用をおよぼし, 細胞膜を損傷させることによって膜透過性が向上することを明らかにした. 損傷発生の頻度の検討から, 膜損傷は微小気泡の存在に強く依存し, 音圧に依存して増加することを示した. さらに, 照射3分後における細胞膜損傷率が照射直後の約30%に低下したことから, 膜修復は非常に速い現象であることが示された. 結語：パルス超音波と微小気泡を用いたsonoporationにおける細胞膜損傷と修復現象の発生と頻度について検討し, 薬物導入効率の向上のための基礎的知見を得た.

Purpose. Sonoporation is an ultrasound technique that enables large molecules that normally do not penetrate the cell membrane to pass through it. Recent studies show that pulsed ultrasound in the presence of microbubbles increases the permeability of the cell membrane. However, the mechanism and basic properties of this sonoporation remain unclear. We thus investigated the mechanism of generation and frequency of occurrence of sonoporation, as well as the repair of a cell membrane damaged by microbubbles. Methods. The spatial relationship between microbubbles and cells was observed microscopically when cells were sonicated with pulsed ultrasound. Effects of microbubbles on the cells were observed with a high-speed camera, and the ratio of cell membrane damage and repair was examined using fluorescent microscopy. Results. Damage to the cell membrane, caused mainly by mechanical effects of the expansion and contraction of microbubbles, significantly increased the permeability of the cell membrane. The frequency of cell membrane damage was closely associated with the presence of microbubbles and increased with increase in acoustic pressure. The ratio of repair of damaged cells was about 70% during 3 min after a single shot of pulsed ultrasound, indicating that repair of damaged cell membranes requires little time. Conclusion. We examined the frequency of occurrence of cell membrane damage and repair in sonoporation using pulsed ultrasound and microbubbles. Our results should prove useful for improving pulsed-ultrasound sonoporation.