不同脉冲超声参数及培养方式对细胞活力和细胞膜声孔作用的影响

Effects of different pulsed ultrasound parameters and culture conditions on cell viability and sonoporation on cell membrane

目的 探寻不同脉冲超声（PUS）辐射策略及细胞培养方式对宫颈癌细胞（Hela）活力和细胞膜声孔作用的影响。方法以2种不同的方式（悬浮培养或贴壁培养）培养Hela细胞，改变声强（0．4W／cm^2、1．0W／cm^2、1．6W／cm^2和2．2W／cm^2）、占空比（10％、20％、50％）以及辐射时间（1min、3min）等参数，对Hela细胞进行辐射，运用流式细胞术行细胞活力分析，应用显微镜和扫描电镜观察细胞形态变化及细胞膜表面的超微结构，比较不同参数组合方案的影响。结果低声强和低占空比辐射未造成明显细胞死亡，高声强（1．6W／cm^2和2．2W／cm^2）和高占空比（50％）辐射显著降低细胞存活率（P〈0．01），导致细胞脱离。析因设计分析可知，细胞培养方式和3种超声参数均有显著的交互作用（P〈0．01），对细胞活力有很大的影响。电镜结果显示，适当条件的超声辐射能导致细胞表面出现可逆性孔道。通过优化可获得合理的平衡，细胞存活率大于80％。悬液培养的细胞经1．0W／cm^2、20％占与空比的PUS辐射3min后，声孔作用最强，出现的孔洞最多。结论对培养方式和超声参数进行优化后可减少细胞损伤，产生较强的声孔作用，增加细胞膜通透性，有利于大分子物质的胞内输送。

Objective To investigate different pulsed ultrasound （PUS） parameters and culture conditions that would affect cell viability and sonoporation on cell membrane of human cervical cancer cells （ HeLa）. Methods HeLa cells were cultured in two different conditions （in suspension or in monolayer）. Cells were exposed to different PUS intensity （0.4 W/cm^2, 1.0 W/cm^2, 1.6 W/cm^2, 2.2 W/cm^2） , duty cycle （10% , 20% , 50% ） and exposure time （ 1 min or 3 min）. Cell viability was analyzed by flow cytometry. Using microscope and scanning electron microscopy （SEM） , the changes of shape and the sonoporation on cell membrane induced by PUS were observed. Results Low intensity and duty cycle did not exert a great impact on the cell viability. Cell injury was found to increase progressively with high intensity （ 1.6 W/cm^2 , 2.2 W/cm^2 ） and duty cycle （50%） （ P 〈 0.01 ） , and cell detachment was significantly accompanied by PUS exposure in adherent HeLa cells. Results of factorial design showed that the culture conditions and the PUS parameters had significant interaction （ P 〈 0.01 ）. SEM demonstrated in some detail the phenomenon of transient pores in the cell membrane under suitable PUS irradiation. The ideal sonoporation conditions that cell viability was above 80% and more membrane holes were noted to be at 1.0 W/cm^2 exposure for 3 min with a duty cycle of 20% in cell suspension. Conclusion The optimized conditions of the PUS parameters and the culture conditions could lower the cell injury and exert a great impact on the sonoporation. It could produce remarkable membrane pores on cells and enhance cell membrane permeability, which facilitate transportation of macromolecules into cells.