正交优化低频低功率超声联合微泡诱导前列腺癌DU145细胞早期凋亡的实验研究

摘要

Objective To optimize the parameters of the low-frequency/low-energy ultrasound combined with micro-bubbles in inducing early apoptosis of DU145 cells (an androgen-independent prostatic cancer cells). Methods In our study, the impact of ultrasonic power, micro-bubbles/cell suspension volume rate and irradiation time were investigated. Three levels of each factor were deifned as ultrasonic power (60, 80, 100 mW), micro-bubbles/cell suspension volume rate (10%, 20%, 30%), irradiation time (30, 60, 90 s). According to the three-factor three-level orthogonal design, nine experiments were carried out. The early apoptosis was detected by lfow cytometry. A new experiment was designed with the optimized parameters. Another group without ultrasound irradiation was designed as the control group. Flow cytometry and transmission electron microscope (TEM) were used to detect the early apoptosis. Results In descending order, the inlfuence of these factors on the cell early apoptosis were:ultrasonic power>micro-bubbles/cell suspension volume rate>irradiation time. Moreover, the inlfuence of each factor level were:80 mW>60 mW>100 mW in ultrasonic power, 20%>30%>10%in micro-bubbles/cell suspension volume rate, 60 s>90 s >30 s in irradiation time. The early apoptosis rate of experiment group was 10.41%, while the control group was 0.94%. TEM showed apoptotic cells in the experiment group. Conclusions The optimized parameter of low-frequency/low-energy ultrasound with micro-bubbles in inducing early apoptosis of DU145 cells are ultrasonic power of 80 mW, micro-bubbles/cell suspension volume rate of 20%, and irradiation time of 60 s. With the optimized parameters, the early apoptosis rate of the experiment group has signiifcant higher than the control group.%目的：采用正交实验设计法，优化低频（20 kHz）低功率超声联合微泡诱导人雄激素非依赖型前列腺癌DU145细胞早期凋亡的条件。方法以超声功率、微泡/细胞悬液体积比及超声辐照时间为正交优化的3个因素，每个因素设定3个水平，分别为超声功率60、80、100 mW，微泡/细胞悬液体积比10%、20%、30%，超声辐照时间30、60、90 s，根据三因素三水平设计正交实验表，得到9个实验组，按相应条件采用连续波辐照，辐照后细胞继续培养24 h，采用流式细胞术检测细胞早期凋亡。应用正交优化得到的最优组合条件超声辐照实验组DU145细胞，对照组细胞不予超声辐照，采用流式细胞术、透射电镜检测、观察实验组及对照组细胞早期凋亡。结果3个因素对细胞早期凋亡的影响程度为：超声功率＞微泡/细胞悬液体积比＞超声辐照时间。各个因素不同水平对细胞早期凋亡的影响程度分别为：超声功率：80 mW＞60 mW＞100 mW，微泡/细胞悬液体积比：20%＞30%＞10%，超声辐照时间：60 s＞90 s＞30 s。最优组合条件下，实验组细胞早期凋亡率为10.41%，对照组细胞早期凋亡率为0.94%。透射电镜下可见实验组细胞较对照组细胞的体积变小变圆，空泡增多，可见明显的凋亡小体形成。结论低频超声联合微泡诱导人雄激素非依赖型前列腺癌DU145细胞早期凋亡的最优组合条件为：超声功率80 mW，微泡/细胞悬液体积比20%，超声辐照时间60 s。在此条件下实验组早期细胞凋亡率与对照组相比有明显差异。