Medical Engineering Program, The University of Hong Kong, Pokfulam, Hong Kong.
Ultrason Sonochem. 2013 Jan;20(1):247-53. doi: 10.1016/j.ultsonch.2012.08.008. Epub 2012 Aug 11.
Sonoporation (membrane perforation via ultrasonic cavitation) is known to be realizable in plant cells on a reversible basis. However, cell viability may concomitantly be affected over the process, and limited knowledge is now available on how such cytotoxic impact comes about. This work has investigated how sonoporation may affect plant cells at a subcellular level and in turn activate programmed cell death (PCD). Tobacco BY-2 cells were used as the plant model, and sonoporation was applied through a microbubble-mediated approach with 100:1 cell-to-bubble ratio, free-field peak rarefaction pressure of either 0.4 or 0.9 MPa, and 1 MHz ultrasound frequency (administered in pulsed standing-wave mode at 10% duty cycle, 1 kHz pulse repetition frequency, and 1 min duration). Fluoroscopy results showed that sonoporated tobacco cells may undergo plasma membrane depolarization and reactive oxygen species elevation (two cellular disruption events closely connected to PCD). It was also found that the mitochondria of sonoporated tobacco cells may lose their outer membrane potential over time (observed using confocal microscopy) and consequently release stores of cytochrome-c proteins (determined by Western Blotting) into the cytoplasm to activate PCD. These findings provide insight into the underlying mechanisms responsible for sonoporation-induced cytotoxicity in plant cells. They should be taken into account when using this membrane perforation approach for gene transfection applications in plant biotechnology.
声孔（通过超声空化进行细胞膜穿孔）在植物细胞中是可实现的，且具有可逆性。然而，在这个过程中细胞活力可能会同时受到影响，目前对于这种细胞毒性影响的产生机制知之甚少。本研究旨在探究声孔作用如何在亚细胞水平上影响植物细胞，并进而激活程序性细胞死亡（PCD）。烟草 BY-2 细胞被用作植物模型，通过 100:1 的细胞-气泡比、0.4 或 0.9 MPa 的自由场峰值稀疏压力和 1 MHz 的超声频率（在脉冲驻波模式下以 10%占空比、1 kHz 脉冲重复频率和 1 分钟持续时间施加）进行声孔处理。荧光透视结果表明，声孔处理后的烟草细胞可能会经历质膜去极化和活性氧物种的升高（这两个细胞破坏事件与 PCD 密切相关）。研究还发现，随着时间的推移，声孔处理后的烟草细胞的线粒体可能会失去它们的外膜电位（通过共聚焦显微镜观察到），并随后将细胞色素 c 蛋白（通过 Western Blotting 确定）释放到细胞质中以激活 PCD。这些发现为声孔作用引起植物细胞毒性的潜在机制提供了深入的了解。在植物生物技术中使用这种膜穿孔方法进行基因转染应用时，应该考虑到这些机制。
