三氧化二砷诱导人卵巢癌耐药细胞系3AO/cDDP细胞凋亡及机制的研究

Apoptosis of drug-resistant human ovarian carcinoma cell line 3AO/cDDP induced by arsenic trioxide and its mechanism

目的 探讨三氧化二砷 (As2 O3 )对人卵巢癌耐药细胞系 3AO/cDDP细胞生长的影响及其机制。方法 采用四甲基偶氮唑蓝 (MTT)法 ,检测不同浓度As2 O3 作用后 ,3AO/cDDP细胞的生长抑制率 ;采用流式细胞技术检测细胞凋亡率 ,细胞周期变化 ,以及Fas、FasL基因表达的变化。所有结果均与人卵巢癌细胞系 3AO细胞相比较 ;采用细胞骨架染色法观察 3AO/cDDP细胞凋亡细胞形态变化 ,通过吖啶橙染色 ,荧光显微镜观察As2 O3 作用后 3AO细胞的形态变化。结果 As2 O3 能明显抑制3AO/cDDP细胞的增殖 ,抑制作用呈时间和剂量依赖性 (P <0 0 5 ) ;在一定浓度范围内 ,3AO/cDDP细胞凋亡率与As2 O3 的浓度和作用时间呈依赖关系 ,诱导凋亡的最适浓度是 3μmol/L ;As2 O3 低浓度时 ,3AO/cDDP细胞周期S期通过受阻 ,高浓度时诱导S期细胞凋亡 ;As2 O3 作用后 ,两种细胞系Fas基因的表达均呈升调节 ,FasL基因的表达无变化 ;与 3AO细胞相比 ,差异均无显著意义 (P >0 0 5 ) ;As2 O3作用后 3AO及 3AO/cDDP形成典型的凋亡小体。结论 As2 O3 通过Fas/FasL系统 ,诱导S期细胞凋亡 。

Objective To explore the effects of arsenic trioxide (As 2O 3) on the growth of drug resistant human epithelial ovarian carcinoma cell line 3AO/cDDP and its possible mechanism Methods Human epithelial ovarian carcinoma cell line 3AO and drug resistant human epithelial ovarian carcinoma cell line 3AO/cDDP were cultured As 2O 3 of different concentrations was added into the media. Cell culture without addition of As 2O 3 was used as control The growth inhibiting rates of 3AO/cDDP cells with various concentrations of As 2O 3 in different time course (24, 48, 72, and 96 hours after) were studied by methyl thiazolyl tetrazolium (MTT) method The apoptosis percentage, cell cycle phase distribution and expression of Fas/FasL gene were estimated by flow cytometry (FCM) The apoptosis phenotype of 3AO/cDDP cells was observed by cytoskeleton dying, and the apoptosis phenotype of 3AO cells was observed by acridine dying under fluorescent microscopy Results 3AO/cDDP cell growing inhibiting rates by As 2O 3 were different significantly in dose dependent and time dependent manners ( P <0 05) Within a certain concentration range, 3AO/cDDP apoptosis inducing rates by As 2O 3 were dose and time dependent, and the most appropriate concentration was 3 0 μmol/L; lower concentrations of As 2O 3 perturbed the cells to progress through S/G 2 phase, while higher concentrations of As 2O 3 selectively induced apoptosis of S phase cells As 2O 3 up regulated Fas gene expression, but did not affect FasL gene expression in both cell lines without significant difference ( P >0 05) Morphological observation indicated that As 2O 3 induced typical apoptotic bodies in 3AO/cDDP and 3AO cells Conclusion As 2O 3 effectively inhibits the proliferation of drug resistant human ovarian carcinoma cell line through up regulating Fas gene expression and inducing apoptosis of cells in S phase