声化学激活原卟啉Ⅸ杀伤艾氏腹水瘤细胞的分子机理

Molecular mechanisms of the killing effects on Ehrlich ascites tumor cells by sono-chemical-activated protoporphyrin Ⅸ

采用频率为2.2MHz、声强为3W/cm2的低强度聚焦超声结合原卟啉Ⅸ对艾氏腹水瘤细胞的损伤进行研究,并初步探讨其作用的生物学机制。分别用荧光分光光度法、激光共聚焦扫描显微镜分析原卟啉Ⅸ在肿瘤细胞内的积聚、定位情况;利用台盼蓝拒染法测定聚焦超声激活原卟啉Ⅸ对细胞存活率的影响;透射电镜观察细胞的超微结构变化;罗丹明123检测线粒体膜电位变化;2′,7′-二氯荧光黄双乙酸盐(2′,7′-dichlorodihydrofluorescin diacetate,DCFH-DA)检测细胞内活性氧(Reactive oxygen species,ROS)水平;硫代巴比妥酸法检测细胞膜脂质过氧化水平的变化。通过研究发现,艾氏腹水瘤细胞对原卟啉Ⅸ有一定的选择性吸收和留作用,原卟啉Ⅸ主要定位于细胞的线粒体中;在超声结合原卟啉Ⅸ作用下,艾氏腹水瘤细胞的存活率明显下降;细胞的超微结构发生明显损伤;线粒体膜电位下降;ROS生成量显著增加;脂质过氧化水平显著增加。研究结果表明:超声激活原卟啉Ⅸ对艾氏腹水瘤细胞有着明显的协同杀伤效应,其间产生的活性氧自由基作用,降低线粒体膜电位,增加膜的脂质过氧化水平,可能是其损伤艾氏腹水瘤细胞的主要因素之一。

We studied the Ehrlich ascetic tumor （EAT） cell line killing effects by ultrasound activating protoporphyrin Ⅸ （PPIX） and its mechamism. The cellular uptake and subcellular localization of protoporphyrin Ⅸ were estimated by fluorescence photometer and laser scanning confocal microscopy （LSCM） respectively. The trypan blue exclusion method was used to analyze the relative viability of EAT cells after ultrasonically activated protoporphyrin Ⅸ. Changes of uhra-microstructure of the treated cells were examined by transmission electron microscopy. Mitochondrial membrane potential （MMP）, intracellular reactive oxygen species （ROS） levels, and the level of lipid peroxidation were determined by Rhodamine 123, 2＇, 7＇- dichlorodihydrofluorescin diacetate （DCFH-DA） and thiobarbituric acid reaction method respectively. Results showed that there is a selective uptake and retention of the PP[X in tumor cells. CLSM revealed that PPⅨ localize mainly in the mitochondria. In the ultrasound plus PPⅨ group, the survival rate of cells was significantly lower. The uhra-microstructural damage of treated cells was the most remarkable, the loss of mitochondria membrane potential was the most noticeable, levels of ROS and lipid peroxides were remarkably increased. These results indicate that there is significant synergistic killing effect with ultrasound activating PPⅨ. The decreased mitochondria membrane potential and the increased level of lipid peroxidation in the ultrasound plus PPⅨ group may be associated with reactive oxygen species. In summary, reactive oxygen species might be involved in mediating the killing effect of EAT ceils in SDT