Bex2抑制神经胶质瘤细胞凋亡及其与JNK/MAPK通路关系的研究

The inhibitory effect of Bex2 on apoptosis of glioma cells and the association with JNK/MAPK pathway

目的研究Bex2对神经胶质瘤细胞凋亡的影响并探讨其作用机制,为胶质瘤的基因治疗提供理论和实验依据。方法 1利用前期构建pEGFP-N1-Bex2真核表达载体,脂质体转染法过表达Bex2后12、24、48、72 h收集U251神经胶质瘤细胞,用流式细胞术检测细胞凋亡率,用免疫印迹法检测Bex2基因的表达及活化型caspase-3、p-JNK、p-c-Jun的变化。2收集临床神经胶质瘤手术标本,通过免疫组织化学检测Bex2、活化型caspase-3、p-JNK、p-c-Jun蛋白的表达,并与正常脑组织比较,分析它们之间的相关关系。结果 1流式细胞术检测结果显示,过表达Bex2后12、24、48、72 h各时间点细胞凋亡率明显低于空载体组(P<0.05),转染48 h组低于其他时间点组(P<0.05)。免疫印迹法检测表明,转染后48 h细胞的活化型caspase-3、p-JNK、p-c-Jun含量较空载体组和空白对照组降低(P<0.05)。2免疫组织化学结果表明,神经胶质瘤组织较正常脑组织中Bex2表达增高,活化型caspase-3、p-JNK、p-c-Jun蛋白低表达(P<0.05)。结论 Bex2在神经胶质瘤组织中高表达,并通过下调JNK/MAPK通路活性抑制胶质瘤细胞凋亡,进而促进肿瘤的发生和发展。

Objective To study the effects of Bex2 on glioma cell apoptosis and the possible mechanisms, thus offering theoretical and experimental basis for the gene therapy of glioma. Methods The previously contrsucted eu-karyotic expression vector, pEGFP-N1-Bex2, was employed in this study. U251 glioma cells transfected by lipofection transfection after 12, 24, 48 and 72 h Bex2 over-expression were harvested. The apoptotic rate was detected by flow cytometry. The expression of Bex2 gene, and the change of cleaved caspase-3, p-JNK and p-c-Jun were assayed by us-ing immunoblotting. The surgical specimens of clinical glioma were sampled. The expression of Bex2, cleaved caspase-3, p-JNK and p-c-Jun was detected by using immunohistochemistry assay and compared with the normal brain tissues. And the correlations between them were analyzed. Results The results of flow cytometry showed that the cell apop-totic rate was significantly decreased at 12, 24, 48 and 72 h after Bex2 over-expression compared with that of empty carrier group （P〈0.05）. Furthermore, the cell apoptotic rate at 48 h was lower than those of any other detected time points （P〈0.05）. According to the detection of immunoblotting, the contents of cleaved caspase-3, p-JNK and p-c-Jun were decreased at 48 h after Bex2 over-expression compared with those of the empty carrier group and empty control group （both P〈0.05）. The results of immunohistochemistry showed that the expression level of Bex2 in glioma tissues was increased comparing with the normal brain tissues （P〈0.05）. However, the expression of cleaved caspase-3, p-JNK and p-c-Jun were attenuated （P〈0.05）. Conclusion Bex2 is highly expressed in glioma tissues and can inhibit the apoptosis of glioma cell by down-regulation of JNK/MAPK pathway activity, therefore promoting the pathogenesis and development of tumor.