基因靶向抑制反义生存素对脑胶质瘤细胞增殖和凋亡的影响

Effect of Survivin targeting on cell proliferation and apoptosis in glioma cell line

目的探讨反义生存素(Survivin)脂质体复合物对脑胶质瘤细胞增殖和凋亡效应的影响及机制,为脑胶质瘤Surviviu基因靶向治疗提供理论依据。方法用脂质体介导Survivin反义寡核苷酸转染脑胶质瘤细胞系U251细胞;Western印迹试验检测Survivin表达水平变化;噻唑蓝 (MTT)法测量细胞生长情况;流式细胞仪检测细胞凋亡率及线粒体膜电位变化;电镜倒置显微镜观察细胞形态学变化;应用激光共聚焦显微镜免疫荧光分析法评价脂质体反义复合物对Survivin 蛋白的影响。结果反义Survivin脂质体复合物能有效下调Survivin表达水平,肿瘤细胞凋亡率随转染时间延长而逐渐递增,并出现凋亡形态学变化。免疫荧光分析中标记有绿色荧光染色的Sur- vivin蛋白分子在未转染的细胞浆中清晰可见,并呈斑点状分布;而在转染细胞中几乎没有发现。结论靶向抑制Survivin基因在脑胶质瘤未来治疗中有良好的应用前景。

Objective To investigate the effects of antisense Survivin-Lipcompound on U251 human glioma cell proliferation and apoptosis. Methods Survivin oligonueleotide （ODN） was transfected into the U251 glioma cells mediated by Lip reagent. The expression of Survivin protein was detected by Western blotting. MTT assay was applied to determine the proliferation of U251 cells. Apoptosis rate and mitoehondrial membrance potential （MMP） were evaluated by flow eytometrie analysis. The morphological changes were assessed by an electron microscope. Laser scanning confocal microscope immunofluorescence analysis was performed to detect the sub-cellular localization of Survivin protein on treated cells and untreated cells. Results Antisense compound efficiently down regulated the Surviving expression. As revealed by gradually increased apoptoeis rate and decreased MMP in a time dependent manner, and their morphological changes, treatment with antisense compound induced apoptosis and inhibited cell growth. Fluororescein isothiocyanate （FITC）-labeled immunofluorascence staining of Survivin dearly showed that Survivin was expressed mainly in the formation of a spotted distribution inside the cytoplasm of untreated cells. Survivin protein molecules were clearly seen in the cytoplasm of the untransfected cells and distribut- ed like spots and almost disappeared in the transfected ceils with morphological changes conforming to the changes of apoptosis. Conclusion Survivin protein is a key molecule connecting proliferation with apoptosis and antisense oligonueleotides targeting Survivin has a bright prospect in the treatment of glioma ceils.