通过抑制自噬作用增强声动力学疗法诱导的肿瘤细胞凋亡研究

探讨声动力疗法诱导S180细胞凋亡过程中自噬现象的发生及其在细胞凋亡中的作用。用频率为1.1 MHz、功率为3 W/cm²的超声，结合1 μg/mL原卟啉Ⅸ作用于S180细胞，MTT检测细胞存活率；DAPI染色检测凋亡细胞核形态变化；Rhodamine 123 染色检测线粒体膜电位；Western blotting检测自噬标记分子LC3由Ⅰ型向Ⅱ型的转化；吖啶橙（AO）活细胞染色观察自噬小体。声动力处理后，S180细胞明显受损，MTT显示存活率为59.3%并且出现了典型的凋亡形态学特征，线粒体膜电位下降至MFI=971.28，LC3-II表达明显升高；利用自噬和凋亡的抑制剂实验结果提示自噬体的形成（1 h）早于细胞凋亡的发生 (8 h)，自噬抑制剂3-MA和Ba A1增强了SDT对线粒体膜电位的破坏，DAPI染色进一步证实了自噬抑制剂有利于促进SDT诱导的S180细胞凋亡。提示自噬参与了SDT诱导的S180细胞死亡，自噬特异性药物抑制剂增强了SDT诱导的细胞凋亡。

Sonodynamic Therapy Enhanced Tumor Cell Apoptosis by Inhibiting Autophagy

This study is aimed to determine whether autophagy occurred aftor SDT, and to investigate its relationship with apoptosis. S180 cells were examined after SDT induction with ultrasound at a frequency of 1.1MHz and a power of 3W with the presence of 1 μg/ml protoporphyrin Ⅸ. MTT assay was used to evaluated cell viability, DAPI staining was employed to examine the nuclear damage during apoptosis, the fluorescent dye rhodamine 123 was used to detect the mitochondrial membrane potential（MMP）, western blot was applied to assess the processing of LC3 Ⅰto LC3 Ⅱ and acriding orange staining was used to identify the formation of acidic vesicle organelles (AVOs). Following SDT treatment, the cell viability was 593% and the apoptotic features such as chromatin condensation and MMP loss (MFI=971.28) were prominent, autophagy was indentified by the increased LC3 II expression. The relationship between autophagy and apoptosis was studied by applying pharmacological inhibition of autophagy or apoptosis. Data indicated that autophagy (1 h) occurred earlier than apoptosis (8 h). The autophagy inhibitors either 3 methyladenine（3 MA）or bafilomycin A1（Ba A1）led to increased dissipation of mitochondrial membrane potential. DAPI staining further confirmed that autophagy inhibitors enhanced SDT induced cell apoptosis. This article illustrated autophagy participated in SDT caused S180 cell death, and autophagy special inhibitors promoted cellular apoptotic response to SDT.