氧化应激对血管内皮细胞中转录因子FoxO1磷酸化的调节作用

发布时间：2017-12-31 01:11

  本文关键词：氧化应激对血管内皮细胞中转录因子FoxO1磷酸化的调节作用　出处：《南京医科大学》2010年硕士论文　论文类型：学位论文

  更多相关文章： 过氧化氢 FoxO1 血管内皮细胞 Akt

【摘要】： 目的: 内皮功能障碍是许多心血管疾病的共同的危险因素。氧化应激引起的细胞损伤是内皮细胞功能障碍的重要机制之一。FoxO(Forkhead box O)是Forkhead转录因子的一个亚家族,它是一类重要的氧化还原敏感的转录因子,参与细胞周期调控、凋亡、氧化应激抵抗等生物学过程。本文采用过氧化氢(H2O2)刺激血管内皮细胞模拟氧化应激模型,探讨H2O2对细胞内转录因子FoxO1的磷酸化调节作用及对其转录活性的影响。 方法: 用贴块法分离培养大鼠主动脉内皮细胞(rat aortic endothelial cells,RAECs),倒置显微镜下观察细胞形态,并用免疫细胞化学法检测内皮细胞表面Ⅷ因子相关抗原和活细胞摄取Dil-Ac-LDL的方法鉴定RAECs纯度。用western blot法检测RAECs和人血管内皮细胞株EA.hy926中FoxO各亚型FoxO1、FoxO3a和FoxO4的表达。亚融合状态的血管内皮细胞经血清饥饿处理24小时后用于实验。为检测不同程度的氧化应激对FoxO1磷酸化的影响,分别以200μmol/L、500μmol/L或1000μmol/L H2O2处理血管内皮细胞20min,收集细胞总蛋白,用western blot法检测FoxO1的S256位点和T24位点的磷酸化水平。选取500μmol/L浓度的H2O2刺激血管内皮细胞20min、30min或60min后,用western blot法检测FoxO1的磷酸化与刺激时间的关系。为明确FoxO1的磷酸化是否与PI3K/Akt信号通路有关,我们检测不同程度和不同作用时间的氧化应激对蛋白激酶Akt磷酸化的影响,同时运用两种特异性的PI3K抑制剂LY294002或wortmannin预先阻断该信号通路,用western blot法观察氧化应激对FoxO1及Akt磷酸化水平的影响。为观察氧化应激引起的磷酸化对FoxO1亚细胞定位的影响,在500μmol/L H2O2刺激RAECs20min后,我们分别提取了细胞浆与细胞核蛋白,用western blot法检测磷酸化FoxO1的分布,并利用免疫细胞化学方法和荧光显微镜观察磷酸化FoxO1的亚细胞定位。为进一步研究氧化应激诱导的FoxO1磷酸化对其转录活性的影响,将RAECs共转染含有FoxO结合序列IRS(insulin-responsive sequence)的荧光素酶报告基因质粒(3×IRS-luc)和FoxO1的表达质粒或对照质粒, 24小时后给予细胞200μmol/L H2O2刺激12小时,裂解细胞,用双荧光素酶报告基因试剂盒测定荧光素酶的活性。最后,我们检测了氧化应激引起的FoxO1磷酸化对其靶基因Bim转录的影响。10μmol/L LY294002预处理RAECs 1h后给予200μmol/L H2O2刺激4h,提取细胞的总RNA,通过Real-time PCR方法检测靶基因Bim的mRNA水平。 结果: 免疫细胞化学和活细胞摄取Dil-Ac-LDL的方法鉴定RAECs纯度为90%以上。RAECs和EA.hy926细胞株内FoxO1及FoxO3a表达丰富。血管内皮细胞在不同浓度H2O2作用20min后,FoxO1的S256位点和T24位点出现磷酸化,其强度呈浓度依赖性;而500μmol/L作用血管内皮细胞后,FoxO1的S256位点和T24位点磷酸化在20min、30min及60min时都能检测到。H2O2刺激对细胞中总FoxO1的含量没有明显影响。Akt的磷酸化呈现与FoxO1相似的浓度和时间依赖性。H2O2诱导的Akt及FoxO1的磷酸化可以被10μmol/L LY294002或500nmmol/L wortmannin预处理所抑制。500μmol/L H2O2作用RAECs20min后,细胞浆组分中磷酸化的FoxO1明显增多,并可以观察到细胞浆中磷酸化FoxO1(T24)荧光的增强。通过报告基因检测发现,FoxO1能明显激活3×IRS-luc报告基因的荧光素酶活性,而H2O2对这一效应有明显的抑制作用。Real-time PCR检测结果显示,Bim mRNA表达水平在磷酸化的影响,同时运用两种特异性的PI3K抑制剂LY294002或wortmannin预先阻断该信号通路,用western blot法观察氧化应激对FoxO1及Akt磷酸化水平的影响。为观察氧化应激引起的磷酸化对FoxO1亚细胞定位的影响,在500μmol/L H2O2刺激RAECs20min后,我们分别提取了细胞浆与细胞核蛋白,用western blot法检测磷酸化FoxO1的分布,并利用免疫细胞化学方法和荧光显微镜观察磷酸化FoxO1的亚细胞定位。为进一步研究氧化应激诱导的FoxO1磷酸化对其转录活性的影响,将RAECs共转染含有FoxO结合序列IRS(insulin-responsive sequence)的荧光素酶报告基因质粒(3×IRS-luc)和FoxO1的表达质粒或对照质粒, 24小时后给予细胞200μmol/L H2O2刺激12小时,裂解细胞,用双荧光素酶报告基因试剂盒测定荧光素酶的活性。最后,我们检测了氧化应激引起的FoxO1磷酸化对其靶基因Bim转录的影响。10μmol/LY294002预处理RAECs 1h后给予200μmol/L H2O2刺激4h,提取细胞的总RNA,通过Real-time PCR方法检测靶基因Bim的mRNA水平。H2O2刺激内皮细胞后显著增高, PI3K抑制剂LY294002预处理则进一步增加了Bim的表达。 结论: 氧化应激引起血管内皮细胞中转录因子FoxO1的S256位点和T24位点磷酸化,其磷酸化水平呈一定的浓度与时间依赖性。上述位点的磷酸化是通过PI3K/Akt信号通路介导的。磷酸化导致FoxO1从细胞核转移至细胞浆,并对其转录活性有负性调控作用。
[Abstract]:Objective:
Endothelial dysfunction is a common risk factor of many cardiovascular diseases. Cell damage caused by oxidative stress is an important mechanism of endothelial cell dysfunction in.FoxO (Forkhead box O) is a superfamily of transcription factor Forkhead, which is a kind of important redox sensitive transcription factors, involved in cell cycle regulation, apoptosis, oxidation stress resistance and other biological processes. This paper uses hydrogen peroxide (H2O2) stimulation of vascular endothelial cells to mimic oxidative stress model, the effect of H2O2 on the intracellular phosphorylation of transcription factor FoxO1 and its effect on regulation of transcription activity.
Method:
Cultured rat aortic endothelial cells by explant separation (rat aortic endothelial cells, RAECs), cell morphology was observed under inverted microscope, and detected by immunocytochemistry on endothelial cell surface associated antigen and live cell uptake of Dil-Ac-LDL method to identify the purity of RAECs. FoxO Western blot and RAECs was detected in human vascular endothelial cells in the strain of EA.hy926 subtype FoxO1, expression of FoxO3a and FoxO4. The sub fusion state of vascular endothelial cells by serum starvation for 24 hours after the experiment. Effects of oxidative stress test in different degree of FoxO1 phosphorylation, respectively 200 mol/L, 500 mol/L or 1000 mol/L H2O2 treatment of vascular endothelial cells 20min collect, total cell protein was detected by FoxO1, Western blot S256 and T24 site of phosphorylation. Select 500 mol/L concentration of H2O2 stimulated endothelial cell 20min, 30min or 60 Min, with the detection of FoxO1 by blot Western phosphorylation and stimulation time. To determine whether FoxO1 phosphorylation of PI3K/Akt pathway, we examine the effects of oxidative stress in different degree and different action time on protein kinase Akt phosphorylation, while the use of two kinds of specific PI3K inhibitor LY294002 or wortmannin in advance blocking this pathway, to observe the effects of oxidative stress on FoxO1 and Akt phosphorylation by Western blot method. To observe the oxidative stress caused by the effects of phosphorylation on the subcellular localization of FoxO1 and H2O2 in 500 mol/L after RAECs20min stimulation, we extracted cytoplasm and nuclear protein distribution using Western blot method to detect phosphorylation FoxO1, and the subcellular localization of immunocytochemistry and fluorescence microscopy to observe the phosphorylation of FoxO1. For the further study of FoxO1 phosphorylation induced by oxidative stress Effect on the transcriptional activity of RAECs co transfected with FoxO binding sequence of IRS (insulin-responsive sequence) luciferase reporter gene plasmid (3 * IRS-luc) FoxO1 expression plasmid and control plasmid or, given 24 h after 200 mol/L stimulation of H2O2 cells for 12 hours, cell lysis, using dual luciferase activity KIT gene determination of luciferase. Finally, we tested the oxidative stress caused by FoxO1 effect of phosphorylation on its target gene transcription of Bim to 200 mol/L H2O2 4h.10 mol/L LY294002 stimulated RAECs 1h after pretreatment, extraction of total RNA cells, by detecting the Bim target gene Real-time PCR method mRNA.
Result:
Immunocytochemistry and live cell uptake of Dil-Ac-LDL method for identification of the purity of RAECs was more than 90%.RAECs and FoxO1 in EA.hy926 cells and the expression of FoxO3a. Vascular endothelial cells in different concentration of H2O2 20min, FoxO1 S256 and T24 site appears phosphorylation, its strength in a concentration dependent manner; vascular endothelial cells 500 mol/L after the action of FoxO1, S256 and T24 site of phosphorylation at 20min, 30min and 60min can be detected by the stimulation of.H2O2 content on the total FoxO1 cells had no obvious effect on Akt and FoxO1 phosphorylation of.Akt and FoxO1 showed similar concentration and time dependently induced.H2O2 phosphorylation can be 10 mol/L LY294002 500nmmol/L or wortmannin pretreatment inhibited by.500 mol/L H2O2 RAECs20min, cytoplasmic components in the phosphorylation of FoxO1 increased significantly, and can be observed in the cytoplasm of FoxO1 phosphate (T24). Light is enhanced by reporter gene assay showed that FoxO1 could significantly activate 3 x IRS-luc reporter gene luciferase activity, and the inhibitory effect of H2O2.Real-time PCR detection results show this effect, the Bim effect of mRNA expression level in phosphorylation, while the use of two kinds of specific PI3K inhibitor LY294002 or wortmannin blocking the signaling pathway, to observe the effect of oxidative stress on FoxO1 and Akt phosphorylation by Western blot method. To observe the oxidative stress caused by the effects of phosphorylation on the subcellular localization of FoxO1 and H2O2 in 500 mol/L after RAECs20min stimulation, we extracted cytoplasm and nuclear protein distribution using Western blot method to detect the phosphorylation of FoxO1 the subcellular localization and using immunocytochemistry and fluorescence microscopy to observe the phosphorylation of FoxO1. For further research on the phosphorylation of FoxO1 induced by oxidative stress Effects of its transcriptional activity, RAECs co transfected with FoxO binding sequence of IRS (insulin-responsive sequence) luciferase reporter gene plasmid (3 * IRS-luc) FoxO1 expression plasmid and control plasmid or, given 24 h after 200 mol/L stimulation of H2O2 cells for 12 hours, cell lysis, using dual luciferase reporter gene luciferase activity assay kit. Finally, we tested the oxidative stress caused by phosphorylation of FoxO1 effect on the transcription of the target gene Bim 200 mol/L H2O2 4H to stimulate RAECs 1H.10 mol/LY294002 pretreatment, extraction of total RNA cells, significantly increased endothelial cells stimulated by detection of Bim target gene Real-time PCR method mRNA level.H2O2, PI3K effect of LY294002 pretreatment and further increased the expression of Bim.
Conclusion:
S256 and T24 site of phosphorylation of transcription factor FoxO1 in vascular endothelial cells induced by oxidative stress, the phosphorylation level showed a concentration and time dependent. The phosphorylation is mediated through PI3K/Akt signaling pathway. FoxO1 phosphorylation resulted from the nucleus transferred to the cytoplasm, and there is a negative role on its transcriptional activity.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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