【作者】 王勇；

【导师】 陈燕；

【作者基本信息】 华中科技大学 ， 内科血液病学， 2008， 博士

【摘要】 第一部分藤黄酸对淋系肿瘤细胞系的细胞增殖及细胞周期的调节作用【目的】研究藤黄酸对人急性T淋巴细胞白血病细胞株Jurkat细胞和人Burkitt淋巴瘤细胞株Raji细胞体外增殖及细胞周期的影响。【方法】采用MTT法检测藤黄酸处理后的Jurkat细胞和Raji细胞的增殖活性,PI染色流式细胞仪检测细胞周期分布,免疫印迹检测藤黄酸对Jurkat细胞和Raji细胞Cyclin D3及Cyclin E蛋白表达的影响。【结果】(1)藤黄酸对Jurkat和Raji细胞具有明显的增殖抑制作用,藤黄酸作用Jurkat细胞24 h,48 h,72 h的半数抑制浓度IC50分别为1.69±0.09,0.98±0.13,0.67±0.12μmol/L,P < 0.01;藤黄酸作用Raji细胞24 h,48 h,72 h的半数抑制浓度IC50分别为1.34±0.06,0.39±0.03,0.30±0.02μmol/L,P < 0.01。(2)藤黄酸呈剂量依赖式作用于Jurkat细胞和Raji细胞使细胞周期阻滞在G0/G1期,其比例分别由41.78%提高到62.72%与40.23%提高到67.30%。(3)藤黄酸以剂量依赖式降低细胞周期蛋白Cyclin D3和Cyclin E的表达。【结论】藤黄酸能抑制Jurkat细胞和Raji细胞增殖,使Jurkat细胞和Raji细胞的细胞周期阻滞于G0/G1期。藤黄酸的细胞周期阻滞作用可能通过Cyclin D3和Cyclin E的下调有关。第二部分藤黄酸对淋系肿瘤细胞系的细胞凋亡及抗凋亡蛋白的调控作用【目的】研究藤黄酸对人急性T淋巴细胞白血病细胞株Jurkat细胞和人Burkitt淋巴瘤细胞株Raji细胞的诱导凋亡及抗凋亡蛋白的调控作用。【方法】采用Annexin-V/PI双标法检测藤黄酸对Jurkat细胞和Raji细胞的诱导凋亡作用,免疫印迹检测藤黄酸对Jurkat细胞和Raji细胞抗凋亡蛋白NF-κB和Bcl-xL蛋白表达的影响。【结果】0,1,2,3μmol/L藤黄酸处理后的Jurkat细胞的早期凋亡率分别为0.95%、2.09%、34.03%和38.30%。藤黄酸处理后的Raji细胞的早期凋亡率分别为4.21%、10.76%、16.13%和24.38%。以0,0.5,1.0,2.0,4.0μmol/L藤黄酸处理Jurkat细胞24h后,0和0.5μmol/L藤黄酸对NF-κB的表达无明显改变P>0.05,1.0、2.0及4.0μmol/L藤黄酸处理组NF-κB的表达随剂量增加而减低P<0.01;Bcl-xL呈剂量依赖性递减P<0.01。以0,0.5,1.0,2.0,4.0μmol/L藤黄酸处理Raji细胞24h后,,0和0.5μmol/L藤黄酸对Bcl-xL的表达无明显改变P>0.05,1.0、2.0及4.0μmol/L藤黄酸处理组Bcl-xL的表达随剂量增加而减低P<0.01;NF-κB呈剂量依赖性递减P<0.01。【结论】藤黄酸能够诱导Jurkat细胞和Raji细胞凋亡,其机制可能部分通过下调抗凋亡蛋白Bcl-xL和NF-κB的表达来实现。第三部分藤黄酸对淋系肿瘤细胞系死亡诱导清理子的调控作用【目的】探讨藤黄酸对人急性T淋巴细胞白血病细胞株Jurkat细胞和人Burkitt淋巴瘤细胞株Raji细胞的抗癌作用的分子机理。【方法】免疫印迹检测藤黄酸对Jurkat细胞和Raji细胞DIO-1、Caspase-3及其裂解产物p17和p20表达的影响。Hoechst33258及免疫荧光化学双标法检测藤黄酸作用下DIO-1核转位现象。【结果】随着藤黄酸浓度的升高,Jurkat细胞和Raji细胞表达DIO-1的量先升高后降低,对应2.0μmol/L藤黄酸的DIO-1的表达量最高,虽然4.0μmol/L藤黄酸作用24h后DIO的表达较2.0μmol/L组明显降低但仍明显高于对照组;随着作用时间的延长,2.0μmol/L藤黄酸处理后,DIO-1表达量递增。pro-Caspase3片段的表达呈上升趋势且出现相应活化的Caspase3片段p17和p20。虽然两个片段在0.5μmol/L藤黄酸处理时即出现,但显著性的增高出现在2μmol/L藤黄酸组,4μmol/L藤黄酸处理组最高。藤黄酸作用Jurkat细胞和Raji细胞后,DIO-1由胞浆转位入胞核。【结论】藤黄酸能上调DIO-1表达及与DIO-1的核转位有关。藤黄酸诱导Jurkat细胞和Raji细胞凋亡可能通过DIO-1介导,并通过Caspase 3活化来实现。更多还原

【Abstract】 PART I Regulation of gambogic acid on cell proliferation and cell cycle of lymphoid neoplasm cell lines【Objective】To explore the Regulation of gambogic acid on cell proliferation and cell cycle of Jurkat cells and Raji cells.【Methods】Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl) -2,5- diphenyl- tetrazolium bromide assay. Cell cycle was exmined by PI staining thorough flow cytometry. Expressions of Cyclin D3 and Cyclin E were detected by western blotting.【Results】GA inhibited the proliferation of Jurkat cells and Raji cells with 50% inhibitory concentration values of 1.69±0.09 (24 h), 0.98±0.13 (48 h), and 0.67±0.12 mmol/L (72 h) and 1.34±0.06 (24 h),0.39±0.03 (48 h),0.30±0.02μmol/L (72 h), respecitvely. In a dose-dependent manner, Gambogic acid arrested cell cycle of Jurkat and Raji cells at G0/G1 phase, the percentage of which increased from 41.78% to 62.72% and from 40.23% to 67.30%, respectively. Gambogic acid downregulated the expression of Cyclin D3 and Cyclin E dose and time dependently. 【Conclusion】Gambogic acid suppresses the proliferation of Jurkat cells and Raji cells and arrested cell cycle of them at G0/G1 phase. Cell cycle arrest may be through downregulation of Cyclin D3 and Cyclin E.Part II Regulation of gambogic acid on anti-apoptotic protein of Jurkat cells and Raji cells【Objective】To study the Regulation of gambogic acid on anti-apoptotic protein of Jurkat cells and Raji cells.【Methods】Annexin V-fluorescein-isothiocyanate/propidium iodide were used to detect apoptosis. Expressions of Bcl-xL and NF-κB were detected by western blotting.【Results】Treated with 0, 1.0, 2.0, and 3.0μmol/L GA for 24 h, percentages of the early apoptotic cells in the whole Jurkat population was from 0.95% and 2.09% to 34.03% and 38.30%, and that of Raja cells from 4.21% and 10.76% to 16.13% and 24.38%. Treated with 0, 0.5, 1.0, 2.0, and 4.0μmol/L GA for 24 h, expression of Bcl-xL and NF-κB was downregulated dose-dependently except Bcl-xL in Jurkat cells by 0.5μmol/L GA and NF-κB in Raji cells by 0.5μmol/L GA. There were no difference in those groups.【Conclusion】Gambogic acid is able to induce the apoptosis of Jurkat cells and Raji cells, which is partly through the downregulation of the expression of antiapoptotic protein, Bcl-xL and NF-κB. Part III Gambogic acid induces death inducer-obliterator 1-mediated apoptosis【Objective】To explore the apoptosis inducing effects of GA on Jurkat cells and Raji cells and its underlying mechanism.【Methods】Western blotting was used to study the expression of DIO-1 and pro-caspase 3, as well as 2 activated subunits: p17 and p20. The subcellular localization of DIO-1 was examined by immunofluorescence and Hoechst33258 staining.【Results】Treated with 0, 0.5, 1.0, 2.0, and 4.0μmol/L GA for 24 h, the expression of DIO-1 increased dose-dependently and then declined, and pro-caspase 3 was cleavage into 2 subunits: p17 and p20. Treated with 2.0μmol/L GA for 0, 2, and 24h, the expression of DIO-1 increased time-dependently. GA induced the translocation of DIO-1 from cytoplasm into the nucleus in Jurkat and Raji cells.【Conclusion】Gambogic acid upregulates the DIO-1 expression and was related to DIO-1 translocation, leading to the apoptosis of Jurkat and Raji cells. The DIO-1 expression is associated with the activation of pro-caspase 3 accompanied with the downregulation of Bcl-xL and NF-kB. DIO-1 triggered early-stage cell death in GA-treated Jurkat cells.更多还原