B细胞淋巴瘤/白血病-2相关X蛋白基因过表达对人脑胶质瘤U251细胞株Smac/DIABLO表达的影响

Effects of B cell lymphoma/leukemia-2 associated X protein gene over-expression on expression of second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI in human glioblastoma U251 cells

目的 观察B细胞淋巴瘤/白血病-2相关X蛋白（bax）基因过表达对人脑胶质瘤U251细胞第二线粒体来源的半胱氨酸天冬氨酸蛋白水解酶激活剂或低等电点的凋亡蛋白抑制剂家族直接结合蛋白（Smac/DIABLO）表达的影响,并探讨其机制.方法 采用脂质体Lipofectamine2000转染,将携带人bax基因的表达质粒转入人脑胶质瘤U251细胞株中,同时设转染pEGFP空载体和未转染的U251细胞为对照.用Western blot法检测3组细胞中bax、Smac/DIABLO蛋白的表达,用免疫荧光法检测Smac/DIABLO蛋白在细胞中的分布.结果 bax蛋白表达水平在转染外源性bax基因组为0.82±0.12,相较于未转染组（0.35±0.05）、转染pEGFP组（0.32±0.09）显著增高（P＜0.05）.Smac/DIABLO蛋白表达在转染bax组为1.09±0.16,相较于未转染组（0.62±0.10）、转染pEGFP组（0.70 ±0.20）表达上调（P＜0.05）.bax、Smac在未转染和转染pEGFP细胞中表达差异无统计学意义（P＞0.05）.免疫荧光显示Smac/DIABLO主要分布于胞质.结论 外源性bax基因转入人脑胶质瘤细胞中并过表达,能够上调Smac/DIABLO蛋白表达.

Objective To explore the effects of B cell lymphoma/leukemia-2 associated X protein （bax） gene over-expression on the expression of second mitochondria-derived activator of caspase （Smac）/direct IAP-binding protein with low pI （DIABLO） in human glioma cell line.Methods The plasmid of expression vector pEGFP-bax was transfected into human glioblastoma U251 cells with lipofectamine2000,and meanwhile the non-transfected cells and the pEGFP-transfected U251 cells were taken as controls.Cellular bax and Smac/DIABLO expression was determined by Western boltting.Cellular distribution of Smac/DIABLO protein was evaluated by immunofluorescence （IF） assays and observed under inverted fluorescence microscope.Results The bax protein expression level in pEGFP-bax-transfected U251 cells was （0.82 ±0.12）,significantly higher than in non-transfected group （0.35 ±0.05） and pEGFP-transfected control group （0.32 ± 0.09,P 〈 0.05）.The expression level of Smac/DIABLO protein in pEGFP-bax-transfected group was （1.09 ± 0.16）,significantly higher than in non-transfected group （0.62 ± 0.10） and pEGFP-transfected control group （0.70 ± 0.20） （P 〈 0.05）.There was no significant difference in the expression of bax and Smac/DIABLO between non-transfected and pEGFP-transfected groups （P 〉0.05）.IF revealed that Smac/DIABLO was mainly localized in the cytoplasm.Conclusion Transfection of extrinsic bax gene results in its over-expression in U251 cells,which could up-regulate the expression of Smac/DIABLO protein.