四嗪二甲酰胺诱导白血病细胞株SHI-1 凋亡及其分子机制探究.docVIP

四嗪二甲酰胺诱导白血病细胞株SHI?1 凋亡及其分子机制探究 　 作者：周永列，吕亚萍，胡惟孝，邱莲女，王文松，刘建栋，吴建国 毕业论文 【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），用流式细胞术检测ZGDHu?1诱导SHI?1细胞凋亡过程中bcl?2、bax、 p53、Fas蛋白和线粒体膜蛋白的表达变化。用RT?PCR观察经ZGDHu?1作用后SHI?1细胞bcl?2、bax、p53基因表达改变；同时利用实时荧光PCR定量检测端粒酶hTERT?mRNA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。 毕业论文 【关键词】; 四嗪二甲酰胺　细胞株　SHI?1　细胞凋亡　线粒体　端粒酶 毕业论文 　 ; Abstract ; The aim of; study was to investigate; the mechanism 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 were used to treat SHI?1 cells; the; apoptosis of; SHI?1; cells was analyzed by; morphology, DNA agarose gel electrophoresis, DNA content detection, Annexin?V/PI and Hoechst33258 labeling method,; the mitochondrial transmembrance potential（ΔΨm） were measured by dihydrorhodamin 123,; and expressions of bcl?2, bax, Fas, p53 and mitochondrial membrane protein were analyzed by flow cytometry,while the bcl?2,bax and p53 gene; were analyzed by RT?PCR. The transcriptional level; of hTERT?mRNA was measured by real?time fluorescence quantitative RT?PCR.; The results showed that after exposure to ZGDHu?1, SHI?1 cells were; induced to apoptosis in; a time?and does?dependent; manner.; SHI?1 cell; apoposis; was; comfirmed; 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; i