摘要
目的研究组织因子(TF)在人胶质母细胞瘤细胞中影响化疗药物敏感性的信号转导机制。方法以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WST法检测阿霉素的细胞毒性。结果人胶质母细胞瘤细胞系U373MG高表达TF而LN-229细胞系中TF表达水平低;阿霉素细胞毒性在U373MG中较弱,IC50为1.67±0.03μg/ml;显著高于LN-229,IC50为0.87±0.13μg/ml(P<0.05)。磷脂酰肌醇-3-激酶(PI3K)信号途径中的Akt在U373MG中呈强活化,而LN-229中活化水平低。在转染TF-pcDNA3真核表达载体的LN-229细胞中,TF获得强制高表达,与FⅦ作用后可观察到Akt的显著激活。TF强制表达的LN-229细胞中,阿霉素作用后存活细胞数显著增多(P<0.05),预先以PI3K/Akt特异性抑制剂LY294002处理则可使阿霉素作用后的存活细胞数降低到对照水平,抵消TF强制表达对阿霉素细胞毒性的抑制。结论人胶质母细胞瘤中TF的异常高表达可减轻阿霉素的细胞毒性,此效应可能与TF与FⅦ相互作用后激活下游PI3K/Akt信号途径有关。TF介导的信号转导可能藉此影响肿瘤细胞对化疗药物的敏感性和预后。
Objective To investigate the signaling mechanism of regulation of tissue factor（TF） on cytotoxicity of doxorubicin in human glioblastoma cells. Methods 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 signaling were examined by Western blotting. The cytotoxicity of doxorubicin was determined by WST assay. Results Human glioblastoma cell line U373MG expressed high level of TF while LN-229 expressed low level of TF. IC50 of doxorubicin in U373MG cells was 1.67 ± 0. 03 μg/ml, significantly higher than that in LN-229 cells（ 0.87 ± 0.13 μg/ml, P 〈 0.05 ）. Activation of Akt was also strong in U373MG cells but weak in LN-229 cells. Enforced TF expression was achieved in LN- 229 cells trasfected with TF- pcDNA3. Incubation of factor Ⅶ （ F Ⅶ ） with enforced TF- expressing LN- 229 cells increased the phosphorylation of Akt. Although enforced TF expression in transfected LN-229 cells significantly increased the survival rate after doxorubicin treatment（P 〈0.05）, the specific PI3K/Akt inhibitor LY294002 reversed this increasing effect on survival rate. Conclusion These results suggested that TF might interact with FⅦ and decrease cytotoxicity of doxorubicin in glioblastoma via activating PI3K/Akt signaling,
出处
《血栓与止血学》
2010年第2期75-78,共4页
Chinese Journal of Thrombosis and Hemostasis
