【摘要】 目的研究组织因子(TF)表达对阿霉素诱导人神经母细胞瘤细胞系 SK-N-MC 细胞凋亡的影响。方法以 Western blotting 法检测 SK-N-MC 细胞 TF 表达。分子生物学方法构建 TFsiRNA-pSUPER 表达载体,以脂质体介导进行基因转染。以水溶性四唑盐(WST)法检测阿霉素细胞毒性。以Western blotting 法检测阿霉素诱导的 caspase-3、多聚 ADP 核糖转移酶(PARP)的活化水平。以 Hochest33342染色,荧光显微镜下检测凋亡细胞。结果①SK-N-MC 细胞高表达 TF;②转染 TF siRNA-pSUPER 表达载体的 SK-N-MC 细胞,TF 表达水平呈剂量依赖性降低;③转染 TFsiRNA-pSUPER 的 SK-N-MC 细胞及转染 pSUPER 质粒的对照细胞分别以不同浓度阿霉素处理24 h,前者与后者相比存活细胞数显著降低;④转染 TFsiRNA-pSUPER 的 SK-N-MC 细胞及转染 pSUPER 质粒的对照细胞分别以阿霉素处理4、8 h,可见前者较后者 caspase-3、PARP 活化增强;⑤转染 TFsiRNA-pSUPER 的 SK-N-MC 细胞与对照细胞相比,经阿霉素处理后的凋亡细胞数显著增多(P<0.05)。结论人神经母细胞瘤 SK-N-MC 细胞中 TF 异常高表达,以 siRNA 特异性下调 TF 表达后,可增强阿霉素细胞毒性及其诱导的肿瘤细胞凋亡。肿瘤细胞中 TF 的异常高表达可能参与阿霉素诱导凋亡的调控。更多还原

【Abstract】 Objective To investigate the regulation of tissue factor(TF)on doxorubicin-induced ap- optosis in human neuroblastoma.Method The expression of TF was examined by Western blotting. TFsiRNA-pSUPER plasmid was constructed by inserting a specific 19-nt silencing sequence targeting TF gene into pSUPER vector.Transfection of TFsiRNA-pSUPER was performed using lipofectamine 2000.The activa- tion of caspase-3 and PARP induced by doxorubicin was tested by Western blotting.The apoptotic cells were stained by Hochest 33342 and counted under fluorescence inverted microscope.Results ①Human neuro- blastoma cell line SK-N-MC expressed high level of TF.②Downregulation of TF expression was achieved by transfection of TFsiRNA-pSUPER into SK-N-MC cells in a dose-dependent manner.③Cleavage of caspase-3 and PARP was increased in transfected SK-N-MC cell with down-regulation of TF.④TFsiRNA treatment at 1 μg/ml for 8 h significantly increased apoptotic cell number in transfected SK-N-MC cells compared to that in nontranfected cells(P<0.05) while exposing to 1 μg/ml doxorubicin for 8 h.Conclusions Downregulation of TF expression by specific siRNA vector could increase the cytotoxicity of doxorubicin and enhance doxorubi- cin-induced apoptosis in human neuroblastoma cells.更多还原