组织因子/因子Ⅶ激活人胶质母细胞瘤细胞PI3K/Akt信号途径调控凋亡的研究

The regulation of apoptosis by tissue factor/FⅦ in glioblastoma via PI3K/Akt signal

目的:研究组织因子(TF)对化疗药物诱导人胶质母细胞瘤细胞凋亡影响的信号转导机制。方法:分子生物学方法构建TF-pcDNA3表达载体,以脂质体介导进行基因转染。以Western Blotting检测TF表达及信号转导途径活化状态。以Western Blotting检测阿霉素诱导的Caspase-3、PARP的裂解。结果:以TF-pcD-NA3真核表达载体转染低表达TF的LN-229细胞,TF表达水平呈剂量依赖性增强。以FⅦ与转染TF的LN-229细胞作用,可观察到磷脂酰肌醇-3-激酶(PI3K)信号途径的Akt的显著激活,呈时间依赖性。在转染了TF-pcDNA3真核表达载体的LN-229细胞中,TF获得强制高表达,而阿霉素诱导的Caspase-3、PARP的裂解减弱,PI3K/Akt特异性抑制剂LY294002则可以抵消TF强制表达对阿霉素诱导凋亡的抑制。结论:人胶质母细胞瘤中TF的异常高表达可抑制阿霉素诱导的肿瘤细胞凋亡,此效应可能与TF与FⅦ相互作用后对下游PI3K/Akt信号的激活有关。

Objective:To investigate role of tissue factor (TF) in regulation of apoptosis in duced by chemotherapeutic reagent on human glioblastoma and its mechanism.Method:TF-pcDNA3 plasmid was constructed by inserting TF-cDNA into pcDNA3 vector. Transfection of TF-pcDNA3 was performed using lipofectamine2000. The expression of TF and activation of PI3K/Akt signal were examined. The expression of Caspase-3 and PARP were tested by Western blot.Result:Human glioblastoma cell line LN-229 was expressed low level of TF. The TF expression was upregulated in LN-229 cells after transfected with TF-pcDNA3. Incubation of factor Ⅶ(FⅦ) with LN-229 cells,the phosphorylation of Akt in LN-299 cells was increased in a time-dependent manner. Although the TF expression in transfected LN-229 cells was decreased when doxorubicin-induced cleavage of Caspase-3 and PARP,the PI3K/Akt inhibitor LY294002 could reverse this effect.Conclusion:TF might interact with FⅦ and regulate doxorubicin-induced apoptosis in glioblastoma via PI3K/Akt signal.