外源性Bax基因过表达对神经母细胞瘤SH-SY5Y细胞株Smac蛋白表达的影响

目的观察Bcl-2相关X蛋白（Bax）基因过表达对人神经母细胞瘤SH-SY5Y细胞第二个线粒体来源的半胱氨酸酶的激活剂（Smac）蛋白表达的影响。方法采用脂质体Lipofectamine 2000将携带人Bax基因的过表达质粒转入SH-SY5Y细胞（转染组）,同时设空载体组和未转染组。通过G-418筛选后获得转入目的基因的细胞克隆。Hoechst33258染色观察凋亡细胞,免疫印迹法法检测细胞中Bax、Smac蛋白表达,免疫荧光染色检测Bax、Smac蛋白在细胞中的分布。结果转染后用G-418持续筛选2周可获得G418抗性的细胞克隆。Smac主要分布于胞质,Bax主要位于细胞核。转染组SH-SY5Y细胞Bax蛋白表达水平（1.067±0.036）较空载体组（0.436±0.035）和未转染组（0.444±0.046）显著增高（P〈0.05）;转染组Smac蛋白表达水平（1.408±0.044）相比空载体组（0.708±0.021）和未转染组（0.748±0.041）显著增高（P〈0.05）;空载体和未转染组细胞Bax和Snac蛋白表达均无显著差异（P〉0.05）。转染组较未转染组细胞凋亡率显著增加（P〈0.05）。结论外源性Bax基因转入人神经母细胞瘤细胞中并稳定过表达,能够上调Smac蛋白表达,促进细胞凋亡。

Effects of Bax gene over-expression on expression of Smac/DIABLO in human neuroblastoma SH-SY5Y cells

Objective To explore the effects of over-expression of Bcl-2-associated X protein（Bax） gene on the expression of Smac/DIABLO in human neuroblastoma（NB） cell line.Methods The human Bax gene was transfected into human NB SH-SY5 Y cells with plasmid, lipofectamine 2000. The human NB SH-SY5 Y cells into which empty-plasmid was transfected and the non-transfected human NB SH-SY5 Y cells were served as controls. The cell clones into which objective genes had been transfected were obtained after being screened with G-418. The morphological changes of apoptotic cells were observed by Hoechst33258 fluorescent staining. Bax and Smac/DIABLO expression were determined by Western blot analysis. The intracellular distribution of Smac/DIABLO protein were evaluated by immunofluorescence（IF） assays.Results The G-418-resistant cell clones were obtained by screening continually with G-418 for 2 weeks after the transfection. Bax levels in Bax-transfected group（1.067±0.036） were significantly higher than those in the empty plasmid-transfected group（0.436 ± 0.035; P〈0.05） and non-transfected group（0.444 ± 0.046; P〈0.05）. The levels of Smac/DIABLO protein in Bax-transfected group（1.408±0.044） were significantly higher than those in the empty plasmid-transfected group（0.708±0.021; P〈0.05） and non-transfected group（0.748±0.041; P〈0.05）. But there was no significance differences in the expressions of Bax and Smac/DIABLO between both the control groups（P〉0.05）. IF assay showed that Smac/DIABLO was mainly localized in the cytoplasm. The typical apoptotic morphological changes were found by Hoechst33258 staining in both the Bax-transfected and non-transfected groups. The apoptotic rate of the cells in Bax-transfected group （19.26±1.11）%] was significantly higher than that in non-transfected group （2.96 ± 0.61）%; P〈0.05].Conclusions Transfection of extrinsic Bax gene may result in its continuous over-expression in human NB SH-SY5 Y cells, in which the expression of Smac/DIA