组织因子表达下调影响阿霉素诱导人胶质母细胞瘤细胞凋亡的研究

目的研究组织因子（TF）对阿霉素诱导人胶质母细胞瘤细胞凋亡的影响。方法以免疫印迹法检测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。以水溶性四唑盐法检测阿霉素的细胞毒性。以印迹法检测阿霉素诱导的Caspase-3、PARP的裂解。以Hochest33342染色荧光显微镜检测凋亡细胞。结果人胶质母细胞瘤细胞系U373MG高表达TF。转染TFsiRNA-pSUPER表达载体的U373MG细胞及对照细胞分别以阿霉素处理,可见转染细胞较对照细胞Caspase-3、PARP的裂解增强。以不同浓度阿霉素处理48h后,可见转染后的U373MG细胞的存活数较对照细胞明显减少（P〈0.05）。以Hochest33342染色检测到转染后的U373MG细胞经阿霉素处理后的凋亡细胞数显著增多（P〈0.05）。结论人胶质母细胞瘤U373MG中TF异常高表达的下调,可增强阿霉素诱导的细胞凋亡。肿瘤细胞中TF的异常高表达可能影响肿瘤细胞对化疗药物的耐受性。

Effect of Tissue Factor Expression on Cells Apoptosis of Human Glioblastoma Induced by Doxorubicin

Objective To investigate the effect of the expression of tissue factor(TF)on cells apoptosis of human glioblastoma induced by adoxorubicin.Methods The expression of TF was determined by Western blotting.TFsiRNA-pSUPER plasmid was constructed by inserting specific 19-nt silencing sequence targeting TF gene into pSUPER vector.Transfection of TFsiRNA-pSUPER was performed by using lipofectamine 2000TM.The cytotoxicity of adoxorubicin was determined by water-soluble tetrazolium salt assay.The cleavages of Caspase-3 and PARP induced by adoxorubicin were determined by Western blotting.The apoptotic cells of human glioblastomas were stained by Hochest 33342 and counted under a fluorescence inverted microscope.Results Human glioblastoma cell line U373MG expressed high level of TF.Knockdown of TF expression was achieved by transfection of TFsiRNA-pSUPER into U373MG cells.Inhibition of TF expression increased the cytotoxicity of adoxorubicin in the transfected U373MG cells.Cleavages of Caspase-3 and PARP are enhanced in the transfected U373MG cell with downregulation of TF expression,and TFsiRNA treatment significantly increased the apoptosis of transfected U373MG cells exposing to adoxorubicin compared to the control cells(P<0.05).Conclusions It is suggested that knockdown of TF expression can enhance adoxorubicin-induced apoptosis of glioblastoma cells.Over-expression of TF in glioblastoma may contributed to glioblastomas resistance to chemotherapy.