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  

ZGDHu-1-Inducing Apoptosis of SHI-1 Leukemia Cells and Its Molecular Mechanism

作　　者：周永列[1] 吕亚萍[2] 胡惟孝[2] 邱莲女[1] 王文松[1] 刘建栋[1] 吴建国[1] 

机构地区：[1]浙江省人民医院中心实验室,杭州310014 [2]浙江工业大学药学院,杭州310014

出　　处：《中国实验血液学杂志》2007年第3期483-489,共7页Journal of Experimental Hematology

基　　金：浙江省科技厅资助项目(编号2003c33019)

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Ho-echst33258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。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 bel-2, bax, Fas, p53 and mitochondrial membrane protein were analyzed by flow cytometry, while the bel-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/bel-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 while ΔΨm reduced. The expression of hTERT-mRNA significantly decreased. It is concluded that ZGDHu-1 can up-regulate the expression of p53, bax and bax/bel-2. The mitochondrial pathway mediated by descent of mitochondrial transmembrance potential may be one of the mechanisms inducing apoptosis by ZGDHu-1, in which Fas gene also participates. Telomerase may be an effective gene target for anti-tumour effect of ZGDHu-1.

关 键 词：四嗪二甲酰胺 细胞株 SHI-1 细胞凋亡 线粒体 端粒酶 

分 类 号：R733.7[医药卫生—肿瘤] R979.1[医药卫生—临床医学]