本文關(guān)鍵詞： ATP 蛋白酶體抑制 細胞死亡 敏感性　出處：《廣州醫(yī)學(xué)院》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 背景與目的 泛素-蛋白酶體系統(tǒng)(Ubiquitin-protesome systerm, UPS)通過介導(dǎo)細胞內(nèi)多種蛋白的降解而參與細胞生命過程的調(diào)節(jié)。蛋白酶體是腫瘤治療的重要靶點,蛋白酶體抑制誘導(dǎo)的細胞死亡具有敏感性差異。本課題組在前期實驗中首次發(fā)現(xiàn)ATP濃度對26S蛋白酶體活性具有雙向調(diào)控作用,并首創(chuàng)26S蛋白酶體活性-ATP濃度模式圖。ATP是內(nèi)源性小分子,動物不同組織的ATP濃度不一樣,推測蛋白酶體抑制誘導(dǎo)的細胞死亡敏感性受細胞內(nèi)ATP濃度調(diào)控。本文旨在證明細胞內(nèi)ATP濃度決定蛋白酶體抑制誘導(dǎo)細胞死亡的敏感性,為細胞內(nèi)ATP濃度調(diào)控蛋白酶體功能提供依據(jù)。 方法與結(jié)果 1.細胞內(nèi)ATP濃度的調(diào)控 選用前期實驗已明確產(chǎn)能途徑的K562細胞(以糖酵解為主,糖的有氧氧化為輔)和H460細胞(以糖的有氧氧化占絕對優(yōu)勢)為研究對象。通過干預(yù)細胞培養(yǎng)基的葡萄糖含量、使用腺苷供能(Adenosine,Ade)、抑制細胞的產(chǎn)能途徑【氧化磷酸化抑制劑寡霉素(Oligmycin,OLIG);糖酵解抑制劑2-脫氧-右葡萄糖(2-Deoxy-D-Glucose,2DG)】等方式進行調(diào)控。前期實驗證明細胞內(nèi)ATP濃度的調(diào)控是可行的。 2.細胞內(nèi)ATP濃度決定蛋白酶體抑制誘導(dǎo)細胞死亡敏感性的研究 2.1調(diào)控細胞內(nèi)ATP濃度對蛋白酶體抑制誘導(dǎo)K562細胞死亡敏感性的影響 2.1.1右糖培養(yǎng)基與無糖培養(yǎng)基對蛋白酶體抑制劑誘導(dǎo)細胞死亡的影響 右旋葡萄糖是細胞內(nèi)ATP的主要來源物質(zhì),正常培養(yǎng)基的葡萄糖是右旋葡萄糖,培養(yǎng)細胞時細胞內(nèi)ATP濃度高,而使用無糖培養(yǎng)基時細胞內(nèi)ATP濃度低。 ①不同濃度的MG132(0～20μM)和MG262(0～1μM)分別在右糖培養(yǎng)基與無糖培養(yǎng)基的細胞作用不同時間,Annexin V-FITC流式細胞術(shù)檢測顯示MG132與MG262在右糖培養(yǎng)基誘導(dǎo)的細胞死亡比無糖培養(yǎng)基細胞嚴重,并呈劑量依賴關(guān)系,MG132 10μM作用12小時細胞死亡率相差24.1%,MG132 20μM作用24小時細胞死亡率相差59.8%,MG262 1μM作用12小時死亡率相差18.2% ②MG262 1μM分別作用于右糖培養(yǎng)基與無糖培養(yǎng)基細胞,應(yīng)用熒光倒置顯微鏡進行細胞形態(tài)學(xué)改變動態(tài)觀察(相差100X),時程12小時。右糖培養(yǎng)基細胞“出芽”、凋亡小體形成、細胞破裂等細胞死亡形態(tài)學(xué)改變明顯,無糖培養(yǎng)基細胞胞膜基本完整結(jié)果說明細胞內(nèi)ATP濃度對蛋白酶體抑制劑誘導(dǎo)的細胞死亡敏感性存在影響,細胞內(nèi)ATP濃度高時蛋白酶體誘導(dǎo)的細胞死亡嚴重。 2.1.2右糖培養(yǎng)基與左糖培養(yǎng)基對蛋白酶體抑制誘導(dǎo)細胞死亡敏感性的影響左旋葡萄糖是右旋葡萄糖的對映異構(gòu)體,不能代謝供能,用于平衡細胞滲透壓。 ①MG132(5μM)、MG262(1μM)分別在右糖培養(yǎng)基與左糖培養(yǎng)基細胞作用9小時、18小時,流式細胞術(shù)檢測顯示MG132與MG262在右糖培養(yǎng)基誘導(dǎo)的細胞死亡比左糖培養(yǎng)基嚴重,藥物作用9小時細胞死亡率相差約6%,藥物作用18小時細胞死亡率相差約30%,兩藥物各自在右糖培養(yǎng)基與無糖培養(yǎng)基的細胞死亡率比較,具有統(tǒng)計學(xué)意差義,p0.01。 ②MG132(5μM)分別在右糖培養(yǎng)基與左糖培養(yǎng)基細胞作用12小時,電子透射顯微鏡細胞超微結(jié)構(gòu)圖像顯示右糖培養(yǎng)基細胞死亡改變明顯:核濃縮,染色質(zhì)呈新月狀、團塊狀聚集在核膜周圍或核碎裂,胞漿空泡化,細胞膜破裂;左糖培養(yǎng)基細胞死亡改變表現(xiàn)為線粒體腫脹,凋亡小體形成,胞膜完整。 上述結(jié)果可排除滲透壓對蛋白酶體抑制劑誘導(dǎo)細胞死亡敏感性的影響,支持細胞內(nèi)ATP濃度高時蛋白酶體抑制誘導(dǎo)的細胞死亡嚴重。 2.1.3 Ade上調(diào)細胞內(nèi)ATP對蛋白酶體抑制誘導(dǎo)細胞死亡敏感性的影響 ①實驗同時采用無糖培養(yǎng)基與右糖培養(yǎng)基,分對照組,Ade組,MG262組,Ade+ MG262組,藥物作用24小時,流式細胞術(shù)檢測顯示,右糖培養(yǎng)基細胞死亡率50.7%～82%,無糖培養(yǎng)基細胞死亡率15.8%～36.3%;Ade+MG262組與MG262組比較,在無糖培養(yǎng)基表現(xiàn)為細胞死亡加重,兩組細胞死亡率比較,差異有統(tǒng)計學(xué)意義,p0.01;在有糖培養(yǎng)基表現(xiàn)為細胞死亡由早期凋亡走向晚期凋亡,細胞死亡百分率基本不變。 ②實驗采用右糖培養(yǎng)基,分對照組,Ade組,MG262組,Ade+MG262組,MG132組、Ade+MG132組,,藥物作用18小時, LDH測定值MG262組431.31 U/L,Ade+MG262組939.3 U/L,MG132組718.85 U/L、Ade+MG132組1098.85 U/L,,Ade+MG132組與MG132組LDH值比較,差異有統(tǒng)計學(xué)意義,p0.01,Ade+MG262組與MG262組LDH值比較,差異有統(tǒng)計學(xué)意義,p0.01。 結(jié)果表明Ade上調(diào)細胞內(nèi)ATP時,蛋白酶體抑制誘導(dǎo)的細胞死亡加重。 2.1.4 OLIG下調(diào)細胞內(nèi)ATP對蛋白酶體抑制誘導(dǎo)細胞死亡敏感性的影響。 ①OLIG在無糖培養(yǎng)基下調(diào)細胞內(nèi)ATP對蛋白酶體抑制誘導(dǎo)細胞死亡的影響實驗采用無糖培養(yǎng)基,分對照組、OLIG組、Ade組、OLIG+Ade組、MG132組、MG132+OLIG組,藥物作用12小時,流式細胞術(shù)檢測顯示OLIG組細胞死亡率約40%,OLIG+Ade組未見細胞死亡,MG132組細胞死亡率35.3%,OLIG+MG132組與OLIG組細胞死亡率基本相同。 ②OLIG在有糖培養(yǎng)基下調(diào)細胞內(nèi)ATP對蛋白酶體抑制誘導(dǎo)細胞死亡的影響實驗采用有糖培養(yǎng)基,分對照組,OLIG組,MG262組,OLIG+MG262組、MG132組,OLIG+MG132組,藥物作用15小時,Annexin V-FITC流式細胞術(shù)檢測顯示,OLIG+MG262組細胞死亡率33.63%,MG262組細胞死亡率63.7%,兩組細胞死亡率比較,差異有統(tǒng)計學(xué)意義,p0.01;OLIG+MG132組細胞死亡以早期凋亡為主,MG132組細胞死亡以晚期凋亡為主。 上述結(jié)果表明OLIG在無糖培養(yǎng)基下調(diào)細胞內(nèi)ATP時,蛋白酶體抑制誘導(dǎo)的細胞死亡敏感性基本無變化;而OLIG在右糖培養(yǎng)基中下調(diào)細胞內(nèi)ATP時,蛋白酶體抑制誘導(dǎo)的細胞死亡減輕。 2.1.5 2DG下調(diào)細胞內(nèi)ATP對蛋白酶體抑制誘導(dǎo)細胞死亡敏感性的影響 實驗采用有糖培養(yǎng)基,分對照組、Ade組、2DG組,2DG+MG132組,2DG+Ade+MG132組,藥物作用12小時,流式細胞術(shù)檢測顯示MG132組細胞死亡率56.15%,2DG+MG132組細胞死亡率24.75%,2DG+MG132組細胞死亡率13.7%,2DG+Ade+MG132組細胞死亡率49.8%。 實驗結(jié)果表明2DG在右糖培養(yǎng)基中下調(diào)細胞內(nèi)ATP時蛋白酶體抑制誘導(dǎo)的細胞死亡減輕,而使用Ade上調(diào)細胞內(nèi)ATP時蛋白酶體抑制誘導(dǎo)的細胞死亡加重,蛋白酶體誘導(dǎo)的細胞死亡敏感性隨ATP濃度的改變而改變。 2.2調(diào)控ATP濃度對蛋白酶體抑制誘導(dǎo)H460細胞死亡敏感性的影響 2.2.1 OLIG下調(diào)細胞內(nèi)ATP濃度對蛋白酶體抑制誘導(dǎo)細胞死亡敏感性的影響 實驗采用右糖培養(yǎng)基,分對照組、OLIG組、MG132組、OLIG+MG132組,藥物作用6小時,流式細胞術(shù)檢測顯示MG132組細胞死亡率10.23%,OLIG+MG132組細胞死亡率30.27%,OLIG+MG132組與MG132組細胞死亡率比較,p0.05。 2.2.2 2DG下調(diào)細胞內(nèi)ATP濃度對蛋白酶體抑制誘導(dǎo)細胞死亡敏感性的影響 ①實驗采用右糖培養(yǎng)基,分對照組、2DG組、MG132組、2DG+MG132組,藥物作用12小時,流式細胞術(shù)檢測顯示MG132組細胞死亡率20.27%,2DG+MG132組細胞死亡率15%%,2DG+MG132組與MG132組細胞死亡率比較,差異有統(tǒng)計學(xué)意義,p0.05。 ②實驗采用右糖培養(yǎng)基,分對照組、2DG組、MG132組、2DG+MG132組,藥物作用12小時, LDH測定值MG132組373U/L, 2DG+MG132組124U/L,2DG+MG132組與MG132組LDH測定值比較,差異有統(tǒng)計學(xué)意義,p0.05。 上述結(jié)果顯示H460細胞內(nèi)ATP下調(diào)時蛋白酶體抑制誘導(dǎo)的細胞死亡變化不一致,H460細胞的產(chǎn)能途徑以有氧氧化占絕對優(yōu)勢,使用OLIG抑制氧化磷酸化時細胞內(nèi)細胞內(nèi)ATP濃度大幅度下調(diào),蛋白酶體抑制誘導(dǎo)的細胞死亡加重,而2DG使H460細胞內(nèi)ATP小幅度下調(diào),蛋白酶體抑制誘導(dǎo)的細胞死亡減輕。 結(jié)論 1.細胞內(nèi)ATP濃度決定蛋白酶體抑制誘導(dǎo)細胞死亡的敏感性。 2.細胞內(nèi)ATP濃度雙向調(diào)控蛋白酶體抑制誘導(dǎo)的細胞死亡。
[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.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R341

【共引文獻】

相關(guān)期刊論文 前3條

1 Naima Hammoudi;Kausar Begam Riaz Ahmed;Celia Garcia-Prieto;;Metabolic alterations in cancer cells and therapeutic implications[J];癌癥;2011年08期

2 喬靜;陳秀華;;腫瘤細胞的“瓦博格效應(yīng)”與抗腫瘤藥物的研發(fā)[J];藥學(xué)進展;2008年04期

3 韓保衛(wèi);劉光輝;董帥軍;韓鴻彬;劉帥峰;許震;王春友;;糖酵解抑制劑對低氧胰腺癌細胞增殖和凋亡的影響[J];中國普通外科雜志;2009年03期

相關(guān)博士學(xué)位論文 前2條

1 金薇娜;細胞酸堿內(nèi)環(huán)境對白血病細胞分化及耐藥的影響及相關(guān)機制研究[D];北京協(xié)和醫(yī)學(xué)院;2011年

2 張樹華;抑制糖酵解途徑對胰腺癌細胞PANC-1生物學(xué)特性的影響及其機制的研究[D];華中科技大學(xué);2009年

相關(guān)碩士學(xué)位論文 前3條

1 范麗霞;靶向內(nèi)質(zhì)網(wǎng)應(yīng)激的小分子調(diào)節(jié)劑的發(fā)現(xiàn)與生物活性研究[D];華東師范大學(xué);2011年

2 沈正海;干擾TIGAR基因增強表阿霉素對HepG2細胞凋亡誘導(dǎo)作用的實驗研究[D];蘇州大學(xué);2010年

3 韓宜男;新型化療藥物對視網(wǎng)膜母細胞瘤的體外藥效實驗[D];復(fù)旦大學(xué);2010年

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本文編號：1471448