组织因子表达影响阿霉素诱导人神经母细胞瘤凋亡的研究

Regulation of Tissue Factor on Doxorubicin- induced Apoptosis in Human Neuriblastoma

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凋亡的影响。方法以Western印迹法检测TF表达,以WST法检测阿霉素的细胞毒性。以Western印迹法检测阿霉素诱导的Caspase-3、PARP的裂解,以Hochest 33342染色,倒置荧光显微镜下检测凋亡细胞。结果人神经母细胞瘤系SH-EP1高表达TF而SK-N-SH细胞系中TF表达水平低。以TF基因表达载体TF-pcDNA 3转染SK-N-SH细胞,显著增强其TF表达水平。以不同浓度阿霉素处理48 h后,可见转染TF-pcDNA 3的SK-N-SH细胞的存活数较对照细胞明显增多。转染TF-pcDNA 3的SK-N- SH细胞及转染pcDNA3质粒的对照细胞分别以阿霉素处理4 h、8 h、24 h,可见转染TF基因的细胞与对照细胞相比, Caspase-3、PARP裂解减弱。以Hochest 33342染色检测到转染TF-pcDNA 3的SK-N-SH细胞经阿霉素处理后的凋亡细胞数为134．33±21．00,与转染pcDNA 3的对照组相比(232．33±8．84)显著减少(P<0．05)。结论TF在人神经母细胞瘤细胞SK-N-SH中的强制表达,可抑制阿霉素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细胞凋亡的调控,影响肿瘤细胞对化疗药物的耐受性和疾病进展。

Objective To investigate the regulation of tissue factor（TF） on chemotherapeutic reagent induced apoptosis in human neuroblastoma. Methods The expression of TF was examined by Western blotting. TF- pcDNA 3 expression vector was transfected by using lipofectamine 2000. The cytotoxicity of doxorubi- cin was determined by WST assay. The activation of Caspase- 3 and PARP induced by doxorubicin were tested by Western blotting. Apoptotic cells were stained by Hochest 33342 and counted under fluorescence inverted microscope. Results Human neuroblastoma cell line SH- EP1 expressed high level of TF while SK-N- SH was with low- TF level. TF- pcDNA 3 plasmid was transfected to SK- N- SH cells and expression of TF significantly increased. Enforced expression of TF decreased the cytotoxicity of doxorubicin in transfected SK- N- SH ceils. Cleavage of Caspase- 3 and PARP, which are the main effectors of doxorubicininduced apoptosis, also impaired in transfected SK- N- SH cells with upregulation of TF. TF expression also decreased the apoptotic cell number in TF-enforced expressing SK-N-SH cells（ 134.33 ±21.00） contrasted to those control ceils transfected with pcDAN 3 （232.33±8.84 ,P 〈 0.05 ）while exposing to 1μg/ml doxorubicin for 24 h. Conclusion These results suggest that aberrant expression of TF could inhibit doxorubicin- induced apoptosis in human neuroblastoma cells. Over- expression of TF might contribute to chemotherapy resistance in neuroblastoma and its progression, at lest in part, by regulating doxorubicin-induced apoptosis.