四嗪二甲酰胺诱导白血病细胞株SHI1 凋亡及其分子机制研究论文.docVIP

　　四嗪二甲酰胺诱导白血病细胞株SHI1 凋亡及其分子机制研究论文 周永列，吕亚萍，胡惟孝，邱莲女，王文松，刘建栋，吴建国 【摘要】 本研究探讨四嗪二甲酰胺（ZGDHu1）诱导白血病细胞株SHI1凋亡作用的机制。以不同浓度的ZGDHu1在体外培养SHI1细胞，用细胞形态学、DNA凝胶电泳、DNA含量及细胞周期分析、AnnexinV/PI双标记和Hoechst33258荧光染色等分析细胞凋亡。用Rh123/PI测定线粒体跨膜电位（ΔΨm）.freelRNA表达的变化。结果表明：ZGDHu1能诱导SHI1细胞凋亡，呈作用时间和剂量的量效关系，出现典型的细胞形态改变，DNA片段化，亚G1峰检出并显著增加；AnnexinV/PI和Hoechst33258荧光染色后细胞发生凋亡的特征性改变。SHI1细胞经不同浓度的ZGDHu1体外培养后，bcl2基因和蛋白有所下调，但主要是bax基因和蛋白上调，导致bax/bcl2比值明显增高， p53基因和蛋白与Fas蛋白表达也上调，均呈剂量依赖性。随ZGDHu1作用浓度的增加，ΔΨm下降，而线粒体膜蛋白表达显著升高，端粒酶hTERTmRNA的表达显著下调。结论：ZGDHu1通过上调p53基因、bax基因和bax/bcl2比值，使线粒体跨膜电位显著下降的线粒体通路是ZGDHu1诱导细胞凋亡的途径之一，Fas也参与其中，端粒酶有可能是其抗肿瘤的作用靶点。 【关键词】 四嗪二甲酰胺 细胞株 SHI1 细胞凋亡 线粒体 端粒酶 Abstract The aim of study echanism of N,N′di(mmethylphenyl)3,6dimethyl 1,4dihydro1,2,4,5tetrazine1,4dicarboamide(ZGDHu1) inducing apoptosis in SHI1 human leukemia cell line. Different concentrations of ZGDHu1 and different times of culture orphology, DNA agarose gel electrophoresis, DNA content detection, AnnexinV/PI and Hoechst33258 labeling method, the mitochondrial transmembrance potential（ΔΨm） easured by dihydrorhodamin 123, and expressions of bcl2,.freelitochondrial membrane protein etry,RNA easured by realtime fluorescence quantitative RTPCR. The results shoeand doesdependent manner. SHI1 cell apoposis ed by typical cell morphology, DNA fragmentation, subG1 phase, Hoechst33258 and AnnexinⅤ/PI labeling etc. The expression of bax, bax/bcl2, p53 and Fas gene significantly increased and bcl2 slightly decreased. ZGDHu1 could increased the expression of mitochondrial membrane protein in a dosedependent manner reduced. The expression of hTERTmRNA significantly decreased. It is concluded that ZGDHu1 can upregulate the expression of p53, bax and bax/bcl2. The mitochondrial pathediated by descent of mitochondrial transmembrance potential may be one of the mechanisms inducing apoptosis by ZGDHu1, in erase may be an effective gene target for antitumour effect of ZGDHu1. Key methylphenyi)3,6