细胞内ATP浓度决定蛋白酶体抑制诱导细胞死亡的敏感性

发布时间：2018-01-28 20:13

本文关键词： ATP 蛋白酶体抑制细胞死亡敏感性　出处：《广州医学院》2009年硕士论文　论文类型：学位论文

【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,p0.01。 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,p0.01;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,p0.01,Ade+MG262组与MG262组LDH值比较,差异有统计学意义,p0.01。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,p0.01;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,p0.05。 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,p0.05。 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,p0.05。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。
[Abstract]:Background and purpose
The ubiquitin proteasome system (Ubiquitin-protesome systerm, UPS) in regulating cellular processes mediated by degradation of proteins within the cell. The proteasome is an important target for tumor therapy, proteasome inhibition induced cell death with different sensitivity. The research group for the first time in the previous experiment showed that ATP concentration has bidirectional regulation the 26S proteasome activity, and the first 26S proteasome activity concentration of -ATP pattern.ATP is an endogenous small molecule, the concentration of ATP in different tissues of the animal is not the same as that of proteasome inhibition induced cell death was regulated by the intracellular concentration of ATP. This thesis aims to prove that the intracellular ATP concentration determines the sensitivity of proteasome inhibition induced cell death and to provide the basis for the concentration of intracellular ATP regulation of proteasome function.
Methods and results
Regulation of ATP concentration in 1. cells
The early experiments have clear capacity approach of K562 cells (in glycolysis and aerobic oxidation of glucose supplement) and H460 cells (by aerobic oxidation of sugar accounted for absolute advantage) as the research object. The content of glucose medium through the intervention of cells, used for adenosine (Adenosine, Ade), can restrain production way [cell oxidative phosphorylation inhibitor oligomycin (Oligmycin, OLIG); glycolysis inhibitor 2- deoxy glucose right (2-Deoxy-D-Glucose, 2DG)] by means of regulation. Preliminary experiments have demonstrated that the regulation of the intracellular concentration of ATP is feasible.
The study of ATP concentration in 2. cells determines the inhibition of proteasome inhibition induced cell death sensitivity
2.1 effects of intracellular ATP concentration on the death sensitivity of K562 cells induced by proteasome inhibition
Effect of 2.1.1 right sugar medium and sugar free medium on cell death induced by proteasome inhibitor
Dextrose is the main source of ATP in cells. Glucose in normal media is dextrose glucose. When cultured cells, the concentration of ATP is high, while the ATP concentration in cells is low when using sugar free medium.
The different concentrations of MG132 (0~20 M) and MG262 (0~1 M) respectively in the right sugar medium and sugar free medium cells for different periods of time, Annexin V-FITC flow cytometry was used to detect MG132 and MG262 in the right cell death induced by sugar culture medium than sugar free cell culture medium and a serious show dose dependent, MG132 10 M 12 hours of cell death was 24.1% MG132, 20 M 24 hours of cell death was 59.8% MG262, 1 M 12 hour mortality rate is 18.2%
The MG262 1 M were applied to the right sugar medium and sugar free cell culture medium, using fluorescence microscope observation of dynamic changes in cell morphology (phase 100X), duration of 12 hours. The right sugar medium cell budding, formation of apoptotic bodies, obvious morphological changes of cell death cell rupture, sugar free culture based on the basic integrity of the cell membrane results showed that intracellular ATP concentration of proteasome inhibitor induced cell death sensitivity effect induced by ATP at high concentrations of proteasome intracellular cell death seriously.
The effects of 2.1.2 dextran glucose medium and left sugar medium on proteasome inhibition and cell death sensitivity were studied. The left glucose is the enantiomer of dextrose glucose, which can not metabolize energy and balance cell osmotic pressure.
MG132 (5 M), MG262 (1 M) respectively in the right and left sugar sugar medium culture medium cell for 9 hours, 18 hours, flow cytometry showed that MG132 and MG262 in the right cell death induced by sugar culture medium than the left sugar medium serious drugs 9 hours of cell death the phase difference of about 6%, the drug 18 hours cell death rate difference of about 30%, two drugs in their respective right sugar medium compared with sugar free medium cell mortality rate, with statistical significance difference, p0.01.
The MG132 (5 M) were cultured and left sugar cultivation effect of basal cells in 12 hours right sugar, electron microscope cell ultrastructure transmission images showed right sugar medium cell death changed significantly: nuclear condensation, chromatin crescent, lumpy aggregation around the nuclear membrane or nuclear fragmentation, cytoplasmic vacuolization the cell membrane rupture; left sugar culture changes of mitochondrial swelling and death basal cells, formation of apoptotic bodies and cell membrane integrity.
The above results could exclude the effect of osmotic pressure on the sensitivity of proteasome inhibitor to cell death, and support the inhibition of cell death caused by proteasome in the high concentration of ATP.
2.1.3 Ade up-regulated the effect of intracellular ATP on cell death sensitivity induced by proteasome inhibition
At the same time, the sugar free medium and right sugar medium experiment was divided into control group, Ade group, MG262 group, Ade+ MG262 group, the drug for 24 hours, flow cytometry showed that, 50.7% ~ right sugar culture medium cell mortality rate was 82%, the sugar free culture medium cell 15.8% ~ Ade+ MG262 group compared the mortality rate 36.3%; with the MG262 group in the sugar free culture medium for cell death increased, compared with two groups of cell death, a statistically significant difference in P0.01; sugar culture medium for cell death by apoptosis early to late apoptosis, cell death was basically unchanged.
The experiment used the right sugar culture medium, were divided into control group, Ade group, MG262 group, Ade+MG262 group, MG132 group, Ade+MG132 group, and drug action for 18 hours, LDH values of MG262 group 431.31 U/L, 939.3 U/L Ade+MG262 group, MG132 group 718.85 U/L, 1098.85 U/L, Ade+MG132 group, Ade+MG132 group and MG132 group LDH comparison was statistically significant difference between P0.01 group and MG262 group, Ade+MG262 LDH value, the difference was statistically significant, p0.01.
The results showed that the increase of cell death induced by proteasome inhibition was increased by Ade up regulation of intracellular ATP.
2.1.4 OLIG reduces the effect of intracellular ATP on the cell death sensitivity induced by proteasome inhibition.
OLIG in sugar free medium reduced intracellular ATP of proteasome inhibition effect of cell death induced by glucose free medium, divided into control group, OLIG group, Ade group, OLIG+Ade group, MG132 group, MG132+OLIG group, the drug for 12 hours, flow cytometry showed that the cells in OLIG group was about 40%. The OLIG+Ade group 35.3% MG132 group, cell death, cell death, OLIG+MG132 group and OLIG group cell mortality rate is basically the same.
The OLIG in glucose medium and the decrease of intracellular ATP on proteasome inhibition effect induced cell death by sugar culture medium, were divided into control group, OLIG group, MG262 group, OLIG+MG262 group, MG132 group, OLIG+MG132 group, drug 15 hours test showed Annexin V-FITC flow cytometry examination, 33.63% group OLIG+MG262 the rate of cell death and 63.7% cells in MG262 group compared with two groups of mortality, cell death, a statistically significant difference between P0.01 group; OLIG+MG132 cell death in early apoptosis, cell death in MG132 group in late apoptosis.
The above results showed that when OLIG was used to downregulate intracellular ATP in sugar free medium, there was no change in the sensitivity of proteasome induced cell death. While OLIG downregulated ATP in the right glucose medium, proteasome inhibited the death of cell death.
The effect of 2.1.5 2DG down regulation of intracellular ATP on cell death sensitivity induced by proteasome inhibition
The sugar culture medium, were divided into control group, Ade group, 2DG group, 2DG+MG132 group, 2DG+Ade+MG132 group, the drug for 12 hours, flow cytometry showed that 56.15% MG132 group 24.75% 2DG+MG132 group the rate of cell death and cell death, cell death 2DG+ 13.7% 2DG+MG132 group, Ade+MG132 group of cell death 49.8%.
The experimental results show that 2DG in the medium reduced intracellular ATP when proteasome inhibition induced cell death and reduce the right sugar culture, while Ade increased the intracellular ATP proteasome inhibition induced cell death and increased sensitivity to change with the concentration of ATP changes in the proteasome induced cell death.
Effect of 2.2 regulation of ATP concentration on the death sensitivity of H460 cells induced by proteasome inhibition
The effect of 2.2.1 OLIG on the inhibition of cell death sensitivity induced by proteasome inhibition by down-regulation of ATP concentration in cells
The experiment was based on the right glucose medium, and divided into control group, OLIG group, MG132 group and OLIG+MG132 group. The drug action lasted for 6 hours. Flow cytometry showed that the cell death rate of MG132 group was 10.23%, the cell death rate of OLIG+MG132 group was 30.27%, and the cell death rate between OLIG+MG132 group and MG132 group was p0.05..
The effect of 2.2.2 2DG on the inhibition of cell death sensitivity induced by proteasome inhibition by down-regulation of ATP concentration in cells
The experiment used the right sugar culture medium, were divided into control group, 2DG group, MG132 group, 2DG+MG132 group, the drug for 12 hours, flow cytometry showed that 20.27% MG132 group 15%% 2DG+MG132 group the rate of cell death and cell death, 2DG+MG132 group and MG132 group of cell death, the difference was statistically significant, p0.05.
(2) the experiment was based on the right glucose medium, and divided into control group, 2DG group, MG132 group and 2DG+MG132 group. The LDH value was 12 hours after drug treatment, and the values in LDH group were 373U/L, 2DG+MG132 group 124U/L, 2DG+MG132 group and MG132 group, the difference between them was statistically significant.
The results showed that H460 cells in ATP down-regulation of proteasome inhibition induced cell death varied capacity approach of H460 cells in the aerobic oxidation of dominant OLIG inhibited oxidative phosphorylation of ATP cells within the cell concentration greatly reduced, proteasome inhibition induced cell death increased, while the 2DG H460 cells. ATP slightly down, proteasome inhibition induced cell death reduced.
conclusion
The concentration of ATP in 1. cells determines the sensitivity of proteasome to inhibit cell death.
The intracellular ATP concentration in 2. cells regulates the inhibition of cell death induced by proteasome.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341

【共引文献】