【作者】 张小燕；

【导师】 刘金保； 董伟华；

【作者基本信息】 广州医学院 ， 病理学与病理生理学， 2009， 硕士

【摘要】 背景与目的泛素-蛋白酶体系统(Ubiquitin-protesome systerm, UPS)通过介导细胞内多种蛋白的降解而参与细胞生命过程的调节。蛋白酶体是肿瘤治疗的重要靶点,蛋白酶体抑制诱导的细胞死亡具有敏感性差异。本课题组在前期实验中首次发现ATP浓度对26S蛋白酶体活性具有双向调控作用,并首创26S蛋白酶体活性-ATP浓度模式图。ATP是内源性小分子,动物不同组织的ATP浓度不一样,推测蛋白酶体抑制诱导的细胞死亡敏感性受细胞内ATP浓度调控。本文旨在证明细胞内ATP浓度决定蛋白酶体抑制诱导细胞死亡的敏感性,为细胞内ATP浓度调控蛋白酶体功能提供依据。方法与结果1.细胞内ATP浓度的调控选用前期实验已明确产能途径的K562细胞(以糖酵解为主,糖的有氧氧化为辅)和H460细胞(以糖的有氧氧化占绝对优势)为研究对象。通过干预细胞培养基的葡萄糖含量、使用腺苷供能(Adenosine,Ade)、抑制细胞的产能途径【氧化磷酸化抑制剂寡霉素(Oligmycin,OLIG);糖酵解抑制剂2-脱氧-右葡萄糖(2-Deoxy-D-Glucose,2DG)】等方式进行调控。前期实验证明细胞内ATP浓度的调控是可行的。2.细胞内ATP浓度决定蛋白酶体抑制诱导细胞死亡敏感性的研究2.1调控细胞内ATP浓度对蛋白酶体抑制诱导K562细胞死亡敏感性的影响2.1.1右糖培养基与无糖培养基对蛋白酶体抑制剂诱导细胞死亡的影响右旋葡萄糖是细胞内ATP的主要来源物质,正常培养基的葡萄糖是右旋葡萄糖,培养细胞时细胞内ATP浓度高,而使用无糖培养基时细胞内ATP浓度低。①不同浓度的MG132(0～20μM)和MG262(0～1μM)分别在右糖培养基与无糖培养基的细胞作用不同时间,Annexin V-FITC流式细胞术检测显示MG132与MG262在右糖培养基诱导的细胞死亡比无糖培养基细胞严重,并呈剂量依赖关系,MG132 10μM作用12小时细胞死亡率相差24.1%,MG132 20μM作用24小时细胞死亡率相差59.8%,MG262 1μM作用12小时死亡率相差18.2%②MG262 1μM分别作用于右糖培养基与无糖培养基细胞,应用荧光倒置显微镜进行细胞形态学改变动态观察(相差100X),时程12小时。右糖培养基细胞“出芽”、凋亡小体形成、细胞破裂等细胞死亡形态学改变明显,无糖培养基细胞胞膜基本完整结果说明细胞内ATP浓度对蛋白酶体抑制剂诱导的细胞死亡敏感性存在影响,细胞内ATP浓度高时蛋白酶体诱导的细胞死亡严重。2.1.2右糖培养基与左糖培养基对蛋白酶体抑制诱导细胞死亡敏感性的影响左旋葡萄糖是右旋葡萄糖的对映异构体,不能代谢供能,用于平衡细胞渗透压。①MG132(5μM)、MG262(1μM)分别在右糖培养基与左糖培养基细胞作用9小时、18小时,流式细胞术检测显示MG132与MG262在右糖培养基诱导的细胞死亡比左糖培养基严重,药物作用9小时细胞死亡率相差约6%,药物作用18小时细胞死亡率相差约30%,两药物各自在右糖培养基与无糖培养基的细胞死亡率比较,具有统计学意差义,p<0.01。②MG132(5μM)分别在右糖培养基与左糖培养基细胞作用12小时,电子透射显微镜细胞超微结构图像显示右糖培养基细胞死亡改变明显:核浓缩,染色质呈新月状、团块状聚集在核膜周围或核碎裂,胞浆空泡化,细胞膜破裂;左糖培养基细胞死亡改变表现为线粒体肿胀,凋亡小体形成,胞膜完整。上述结果可排除渗透压对蛋白酶体抑制剂诱导细胞死亡敏感性的影响,支持细胞内ATP浓度高时蛋白酶体抑制诱导的细胞死亡严重。2.1.3 Ade上调细胞内ATP对蛋白酶体抑制诱导细胞死亡敏感性的影响①实验同时采用无糖培养基与右糖培养基,分对照组,Ade组,MG262组,Ade+ MG262组,药物作用24小时,流式细胞术检测显示,右糖培养基细胞死亡率50.7%～82%,无糖培养基细胞死亡率15.8%～36.3%;Ade+MG262组与MG262组比较,在无糖培养基表现为细胞死亡加重,两组细胞死亡率比较,差异有统计学意义,p<0.01;在有糖培养基表现为细胞死亡由早期凋亡走向晚期凋亡,细胞死亡百分率基本不变。②实验采用右糖培养基,分对照组,Ade组,MG262组,Ade+MG262组,MG132组、Ade+MG132组,,药物作用18小时, LDH测定值MG262组431.31 U/L,Ade+MG262组939.3 U/L,MG132组718.85 U/L、Ade+MG132组1098.85 U/L,,Ade+MG132组与MG132组LDH值比较,差异有统计学意义,p<0.01,Ade+MG262组与MG262组LDH值比较,差异有统计学意义,p<0.01。结果表明Ade上调细胞内ATP时,蛋白酶体抑制诱导的细胞死亡加重。2.1.4 OLIG下调细胞内ATP对蛋白酶体抑制诱导细胞死亡敏感性的影响。①OLIG在无糖培养基下调细胞内ATP对蛋白酶体抑制诱导细胞死亡的影响实验采用无糖培养基,分对照组、OLIG组、Ade组、OLIG+Ade组、MG132组、MG132+OLIG组,药物作用12小时,流式细胞术检测显示OLIG组细胞死亡率约40%,OLIG+Ade组未见细胞死亡,MG132组细胞死亡率35.3%,OLIG+MG132组与OLIG组细胞死亡率基本相同。②OLIG在有糖培养基下调细胞内ATP对蛋白酶体抑制诱导细胞死亡的影响实验采用有糖培养基,分对照组,OLIG组,MG262组,OLIG+MG262组、MG132组,OLIG+MG132组,药物作用15小时,Annexin V-FITC流式细胞术检测显示,OLIG+MG262组细胞死亡率33.63%,MG262组细胞死亡率63.7%,两组细胞死亡率比较,差异有统计学意义,p<0.01;OLIG+MG132组细胞死亡以早期凋亡为主,MG132组细胞死亡以晚期凋亡为主。上述结果表明OLIG在无糖培养基下调细胞内ATP时,蛋白酶体抑制诱导的细胞死亡敏感性基本无变化;而OLIG在右糖培养基中下调细胞内ATP时,蛋白酶体抑制诱导的细胞死亡减轻。2.1.5 2DG下调细胞内ATP对蛋白酶体抑制诱导细胞死亡敏感性的影响实验采用有糖培养基,分对照组、Ade组、2DG组,2DG+MG132组,2DG+Ade+MG132组,药物作用12小时,流式细胞术检测显示MG132组细胞死亡率56.15%,2DG+MG132组细胞死亡率24.75%,2DG+MG132组细胞死亡率13.7%,2DG+Ade+MG132组细胞死亡率49.8%。实验结果表明2DG在右糖培养基中下调细胞内ATP时蛋白酶体抑制诱导的细胞死亡减轻,而使用Ade上调细胞内ATP时蛋白酶体抑制诱导的细胞死亡加重,蛋白酶体诱导的细胞死亡敏感性随ATP浓度的改变而改变。2.2调控ATP浓度对蛋白酶体抑制诱导H460细胞死亡敏感性的影响2.2.1 OLIG下调细胞内ATP浓度对蛋白酶体抑制诱导细胞死亡敏感性的影响实验采用右糖培养基,分对照组、OLIG组、MG132组、OLIG+MG132组,药物作用6小时,流式细胞术检测显示MG132组细胞死亡率10.23%,OLIG+MG132组细胞死亡率30.27%,OLIG+MG132组与MG132组细胞死亡率比较,p<0.05。2.2.2 2DG下调细胞内ATP浓度对蛋白酶体抑制诱导细胞死亡敏感性的影响①实验采用右糖培养基,分对照组、2DG组、MG132组、2DG+MG132组,药物作用12小时,流式细胞术检测显示MG132组细胞死亡率20.27%,2DG+MG132组细胞死亡率15%%,2DG+MG132组与MG132组细胞死亡率比较,差异有统计学意义,p<0.05。②实验采用右糖培养基,分对照组、2DG组、MG132组、2DG+MG132组,药物作用12小时, LDH测定值MG132组373U/L, 2DG+MG132组124U/L,2DG+MG132组与MG132组LDH测定值比较,差异有统计学意义,p<0.05。上述结果显示H460细胞内ATP下调时蛋白酶体抑制诱导的细胞死亡变化不一致,H460细胞的产能途径以有氧氧化占绝对优势,使用OLIG抑制氧化磷酸化时细胞内细胞内ATP浓度大幅度下调,蛋白酶体抑制诱导的细胞死亡加重,而2DG使H460细胞内ATP小幅度下调,蛋白酶体抑制诱导的细胞死亡减轻。结论1.细胞内ATP浓度决定蛋白酶体抑制诱导细胞死亡的敏感性。2.细胞内ATP浓度双向调控蛋白酶体抑制诱导的细胞死亡。更多还原

【Abstract】 Background and Objective: The ubiquitin-proteasome system (UPS) regulates the vital process of cell by degradation of a variety of proteins, and proteasome is an important target for anti-cancer, the sensitivity of cell death induced by proteasome inhibition is different. After our research group has discovered for the first time that the 26S proteasome activity is regulated bidirectionally by ATP concentrationins in vitro, the 26S Proteasome Activity-ATP Concentration Model is then created. ATP is a kind of endogenous small molecule, and different organs in animal have different ATP concentrations. Therefor, it’s assumed that the sensitivity of cell death induced by protesome inhibition may be regulated by intracellular ATP concentration. The aim of this work is to prove that the intracellular ATP concentration can determine the sensitivity of cell death induced-by proteasome inhibition, thus providing evidencs for the regulation of proteasome function by intracellular ATP concentration.Methods and Results:1. The regulation of the intracellular ATPK562 cell and H460 cell were chosen because their pathway of ATP generation had been disclosed by our previous works. ATP generation in K562 is mainly by glycolytic pathway and partially by aerobic oxidation, while that in H460 is dominantly by aerobic oxidation. Various glucose contents, Adenosine (Ade), Oligomycin (OLIG), 2-Deoxy-D-Glucose (2DG) were used to regulate the intracellular ATP concentration. OLIG is an oxidative phosphorylation inhibitor that blocking mitochondrial/glycolytic ATP generation, and 2DG is aglycolytic inhibitor that blocking glycolytic pathway. Previou works had proved that the intracellular ATP concentration can be control.2. The study of effects intracellular ATP concentration on the sensitivity of cell death induced by proteasome inhibition.2.1 The effects on sensitivity of cell death induced by proteasome inhibition.in K562 by regulating the intracellular ATP concentration.2.1.1 The effects on the sensitivity of cell death induced by proteasome inhibition between culture mediums with D-glucose and without glucoseD-glucose in normal culture medium, is the the main source of intracellular ATP. Thus intrcellular ATP concentration in medium with D-glucose is higher than without glucose.①Different concentrations of MG132 (0 ~ 20μM) and MG262 (0 ~ 1μM) were respectively added to cells in culture medium with D-glucose and without glucose for different hours. Then Annexin V-FITC flow cytometry showed that in D-glucose the cell death induced by MG132 or MG262 was more serious than in no glucose, and was in a dose-dependent manner, the difference of cell death rate being 24.8% with MG132 (10μM) for 12 hours, 59.8% with MG132 (20μM) for 24 hours, and 59.8% with MG262 (1μM) for 12 hours.②MG262(1μM) was respectively added to the cells in culture medium with D-glucose and withou glucose, and then inverted fluorescence microscope was used to observe the changes in cell morphology (ph 100X) dynamically for 12 hours. Cells in culture medium with D-glucose showed significant morphological changes of cell death, such as "budding", apoptotic body formation and cell rupture, in glucose-free medium the integrity of cell membrane were reserved.Therefore, it’s suggested that when intracellular ATP concentration is higher,the cell death induced-by protesome inhibition will be more serious.2.1.2 The effects on sensitivity of cell death induced by proteasome inhibition between culture medium with D-glucose and L-glucoseL-glucose, the enantiomer of D-glucose, cannot generate energy. It was used for the balance of medium osmotic pressure. ①MG132 (5μM) and MG262 (1μM) were respectively added to D-glucose culture mediums with D-glucose and L-glucose for 9 hours and 18 hours. By flow cytometry, it was shown that the cell death in culture medium with D-glucose was more serious than that in culture medium with L-glucose. Difference of cell death rate was about 6% for 9h, about 30% for 18 hours, being statistically significant (p <0.01).②MG132 (5μM) was added to culture mediums with D-glucose and L-glucose 12 hours, and transmission electron microscope (TEM) was used to observe the changes in cell ultrastructure, serious cell death changes in medium with D-glucose, including nuclear enrichment, chromatin presented as crescent-shaped, or clumping and gathered in the nuclear membrane, nuclear fragmentation, cytoplasmic vacuolization and cell membrane rupture; while cells in medium with L-glucose showed slight lethal changes: such as apoptotic bodies, a few cytoplasmic vacuoles and plasma membrane kept integrity.Base on these results, the effect of medium osmotic pressure on cell death could be excluded, and therefore it could be concluded higher intracellular ATP concentration might cause more serious cell death induced by proteasome inhibition.2.1.3 The effects on sensitivity of cell death induced by proteasome inhibition by using Ade to upregulate intracellular ATP①Experiments were performed both in medium with D-glucose and withou glucose,and cells were divided into control group, Adegroup, MG262 group and Ade + MG262 group, with each drug added for 24 hours. Flow cytometry showed that the cell death rate in medium with D-glucose was 50.7% ~ 82%, while 15.8% ~ 36.3% in medium withou glucose. In glucose-free mediu, cell death rate was higher in Ade + MG262 group than MG262 group (p<0.01),while in D-glucose medium, the cell death rates were the same, accompanied by images of varing from early apoptosis to late apoptosis.②Experiment were performed in D-glucose culture medium .Cells were divided into control group, Ade group, MG262 group, Ade + MG262 group, MG132 group and Ade + MG132 group, with each drug added for 18 hours, The LDH values were 431.31 U / L in MG262 group, 939.3 U / L in Ade + MG262group, 718.85 U / L in MG132 group, 1098.85 U / L in Ade + MG132 group. Differences of LDH values between Ade + MG132 group and MG132 group, and between Ade + MG262 group and MG262 group were statistically significant (p <0.01).It is implied that when Ade upregulate the intracellular ATP, cell death induced by proteasome inhibition is more serious.2.1.4 The effect on sensitivity of cell death induced by proteasome inhibition by using OLIG to downregulate intracellular ATP①The effect on sensitivity of cell death induced by proteasome inhibition by using OLIG to downregulate intracellular ATP in glucose-free medium.Experiment was performed in glucose-free medium. Cells were divided into control group, OLIG group, Ade group, OLIG + Ade group, MG132 group, MG132 + OLIG group , with each drug were for 12 hours. By flow cytometry, it was shown that cell death rate in OLIG group was about 40 %, about zero in OLIG + Ade group, and 35.3% in MG132 group, the cell death rate in OLIG + MG132 group was the same as OLIG group.②The effect on sensitivity of cell death induced by proteasome inhibition by using OLIG to downregulate intracellular ATP in D-glucose medium.Experiment was performed with D-glucose medium, cells were divide into control group, OLIG group, MG262 group, OLIG + MG262 group, MG132 group and OLIG + MG132 group, with each drug added for 15 hours. By flow cytometry, it was shown that the cell death rate was 33.63% in OLIG + MG262 group, 63.7% in MG262 group, the difference between the two groups was statistically significant (p <0.01). Images of cell death in OLIG + MG132 group was mainly in early apoptosis, while in MG132 group mainly in late apoptosis.It is implied that downregulating intracellular ATP by OLIG in glucose-free had no effect on the sensitivity of cell death induced by proteasome inhibition, while in D-glucose medium had alleviated the cell death.2.1.5 The effect on sensitivity of cell death induced by proteasome inhibition by using 2DG to downregulate the intracellular ATPExperiment was performed in D-glucose medium. Cells were divided to control group, Ade Group, 2DG group, 2DG + MG132 group and 2DG + Ade + MG132 group , with each drug added for 12 hours. By flow cytometry, it was shown that cell death was 56.15% in MG132 group, 24.75% in 2DG + MG132 group, 13.7% in 2DG + MG132 group, and 49.8% in 2DG + Ade + MG132 group.It was there implied that downregulating the intracellular ATP in D-glucose medium by 2DG can reduce cell death induced by proteasome inhibition, while upwnregulating the intracellular ATP in D-glucose medium by Ade can increase the cell death. The sensitivity of cell death induced by proteasome inhibition was different accompanied by the different intracellular ATP concentration.2.2 The effect on sensitivity of cell death induced by proteasome inhibition in H460 by regulating the intracellular ATP concentration2.2.1 The effect on sensitivity of cell death induced by proteasome inhibition by using OLIG to downregulate the intracellular ATP in D-glucose mediumExperiments were perfoemed in D-glucose medium. Cells were divided into control group, OLIG group, MG132 group and OLIG + MG132 group, with each drug added for 6 hours. By flow cytometry, it was shown that the cell death rate was10.23% in MG132 group, 30.27% in OLIG + MG132 group. There was significant difference between the two group cell death rate (p<0.05).2.2.2 the effect on sensitivity of cell death induced by proteasome inhibition by using 2DG to downregulate the intracellular ATP①Experiments were performed in D-glucose medium. Cells were divided into control group, 2DG group, MG132 group and 2DG + MG132 group, with each drug added for 12 hours. By flow cytometry, it was shown that the cell death rate was 20.27% in MG132 group, 15% in 2DG + MG132 group. There was significant difference between the two group cell death rate (p<0.05).②Experiments were performed in D-glucose medium. Cells were divided into control group, 2DG group, MG132 group and 2DG + MG132 group, with each drug added for 12 hours. The LDH values were 373U / L in MG132 group, 124U / L in 2DG + MG132 group. Difference of LDH values between 2DG + MG132 group and MG132 group was statistically significant (p <0.05). It was shown inconsistent changes existed when downregulating intracellular ATP, that is,since the ATP generation pathway of H460 is dominantly by aerobic oxidation, the intracellular ATP concentration was significantly lower when OLIG was used to block oxidative phosphorylation, resulting in increased cell death induced by protesome inhibition; but 2DG slightly lowered the ATP concentration, and cell death induced by proteasome inhibition was reduced.Conclusions:1. Intracellular ATP concentration determines the sensitivity of cell death induced by proteasome inhibition.2. Intracellular ATP bidirectionally regulates proteasome inhibition-induced cell death.更多还原