Transient Transmembrane Release of Green Fluorescent Proteins With Sonoporation
Transient Transmembrane Release of Green Fluorescent Proteins With Sonoporation
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DOI：
10.1109/tuffc.2010.1586
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发表时间：
2010-07-01
影响因子：
3.6
通讯作者：
Bouakaz, Ayache
中科院分区：
文献类型：
--
作者：
Kaddur, Kadija;Lebegue, Loic;Bouakaz, Ayache
超声（US）激活下的微泡被认为会诱导质膜上的孔形成，导致其渗透，从而导致细胞外环境中的分子掺入。在这项研究中，我们研究了在US和微泡的共同作用下，这种渗透是否也会导致小分子从哺乳动物真核细胞的细胞质中短暂释放。利用稳定表达增强型绿色荧光蛋白（EGFP）基因的Hela细胞，通过流式细胞术测定EGFP阳性细胞（EGFP +细胞）的百分比和细胞平均荧光强度（MFI）来评估EGFP的释放。声波谐振频率为1 MHz，峰值负压范围为0.2至0.6 MPa，占空比为40%和75%，重复频率为10 kHz。结果表明，Hela-EGFP细胞在峰值负压400 kPa和75%占空比条件下，在微泡存在下照射2 min， EGFP+细胞百分比和MFI均下降60%。我们的结果表明，细胞荧光的减少归因于EGFP的释放。最重要的是，这种EGFP释放不是由于超声的致死效应，因为在超声48小时后EGFP表达显著恢复。总之，这项研究首次证明了由声穿孔过程产生的细胞内分子的瞬时释放。这种控制释放显示了通过膜从细胞质中提取分子同时保持细胞活力的可能性。综上所述，本研究的结果加强了声穿孔诱导的瞬态孔隙形成机制的假设。
Microbubbles under ultrasound (US) activation are assumed to induce pore formation in the plasma membrane, causing its permeabilization and hence molecule incorporation from the extracellular environment. In this study, we investigated whether this permeabilization also engenders a transient release of small molecules from the cytosol of mammalian eukaryotic cells under the combined action of US and microbubbles. Using Hela cells stably expressing the enhanced green fluorescent protein (EGFP) gene, the release of EGFP was evaluated by flow cytometry in terms of the percentage of EGFP-positive cells (EGFP + cells) and the mean cell fluorescence intensity (MFI). Sonoporation was performed at 1 MHz, with peak negative pressures ranging from 0.2 to 0.6 MPa, duty cycles of 40% and 75% and a repetition rate of 10 kHz. The results showed that the insonation of Hela-EGFP cells at the peak negative pressure 400 kPa and the 75% duty cycle for 2 min in the presence of microbubbles induced a 60% decrease in both EGFP+ cells percentage and MFI. Our results demonstrate that the reduction of cell fluorescence is attributed to the EGFP release. Most importantly, this EGFP release was not due to lethal effects of sonoporation because the EGFP expression was significantly recovered by 48-h post-insonation. In conclusion, this study demonstrates for the first time a transient release of intracellular molecules produced by the sonoporation process. This controlled release showed the possibility of extracting molecules from the cell cytoplasm through the membrane while preserving cell viability. Taken together, the results obtained in this study reinforce the hypothesis of the transient pore formation mechanism induced by sonoporation.