Wang Pan, Wang Xiao Bing, Liu Quan Hong, Tang Wei, Li Tao
College of Life Sciences, Shaanxi Normal University, Xi'an, China.
Chemotherapy. 2008;54(5):364-71. doi: 10.1159/000151704. Epub 2008 Aug 30.
It was the aim of this study to investigate the ultrasonically induced cytotoxic effect of hematoporphyrin (Hp) on isolated sarcoma 180 (S180) cells and to explore the potential biological mechanism of this action. S180 tumor cells suspended in air-saturated phosphate-buffered saline (pH 7.2) were exposed to ultrasound at 1.6 MHz in a standing wave mode for up to 90 s with and without 100 microg/ml Hp. The intracellular Hp concentration was evaluated to determine the optimum timing of ultrasound exposure after its administration with a fluorescence spectrophotometer based on the standard curve. Cell viability was determined by the trypan blue exclusion test. The morphological changes of S180 cells induced by ultrasonic irradiation were evaluated by scanning electron microscope and transmission electron microscope observation. The participation of lipid peroxidation products in the cell damage process was investigated by the malon aldehyde content test. Our experiments suggested that an incubation time of 45 min after the addition of 100 microg/ml Hp was to be chosen as the best time for ultrasound exposure in vitro. The rate of ultrasonically induced cell damage was enhanced by the increase in ultrasound intensity and exposure time in the presence and absence of Hp. At an ultrasound intensity not less than 3 W/cm(2) and an irradiation time not less than 60 s, a significant synergistic effect of ultrasound combined with Hp was observed, while no cell damage was observed with 100 microg/ml Hp alone. The malon aldehyde content test showed that the lipid peroxidation level significantly increased after sonodynamic therapy treatment. Scanning electron microscope and transmission electron microscope observation indicated that the changes in cell ultra-structure such as cell membrane destruction, mitochondria swelling and chromatin condensation are important factors that induced cell damage in S180 tumor cells. The results indicate that the biological mechanism might be involved in mediating the killing effect on S180 cells, and the ultra-structural changes may account for cell destruction induced by sonodynamic therapy treatment in our experiment mode.
本研究旨在探讨血卟啉（Hp）超声诱导的对分离的肉瘤180（S180）细胞的细胞毒性作用，并探索该作用的潜在生物学机制。将悬浮于空气饱和的磷酸盐缓冲盐水（pH 7.2）中的S180肿瘤细胞，在有和没有100μg/ml Hp的情况下，以驻波模式在1.6 MHz频率下暴露于超声长达90秒。基于标准曲线，用荧光分光光度计评估细胞内Hp浓度，以确定给药后超声暴露的最佳时间。通过台盼蓝排斥试验测定细胞活力。通过扫描电子显微镜和透射电子显微镜观察评估超声照射诱导的S180细胞的形态变化。通过丙二醛含量试验研究脂质过氧化产物在细胞损伤过程中的参与情况。我们的实验表明，添加100μg/ml Hp后45分钟的孵育时间应选为体外超声暴露的最佳时间。在有和没有Hp的情况下，超声诱导的细胞损伤率随着超声强度和暴露时间的增加而提高。在超声强度不低于3 W/cm²且照射时间不低于60秒时，观察到超声与Hp有显著的协同作用，而单独使用100μg/ml Hp未观察到细胞损伤。丙二醛含量试验表明，声动力疗法治疗后脂质过氧化水平显著升高。扫描电子显微镜和透射电子显微镜观察表明，细胞膜破坏、线粒体肿胀和染色质凝聚等细胞超微结构变化是诱导S180肿瘤细胞损伤的重要因素。结果表明，生物学机制可能参与介导对S180细胞的杀伤作用，并且在我们的实验模式下，超微结构变化可能是声动力疗法治疗诱导细胞破坏的原因。
