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  本文關鍵詞：氧化應激對血管內皮細胞中轉錄因子FoxO1磷酸化的調節(jié)作用　出處：《南京醫(yī)科大學》2010年碩士論文　論文類型：學位論文

  更多相關文章： 過氧化氫 FoxO1 血管內皮細胞 Akt

【摘要】： 目的: 內皮功能障礙是許多心血管疾病的共同的危險因素。氧化應激引起的細胞損傷是內皮細胞功能障礙的重要機制之一。FoxO(Forkhead box O)是Forkhead轉錄因子的一個亞家族,它是一類重要的氧化還原敏感的轉錄因子,參與細胞周期調控、凋亡、氧化應激抵抗等生物學過程。本文采用過氧化氫(H2O2)刺激血管內皮細胞模擬氧化應激模型,探討H2O2對細胞內轉錄因子FoxO1的磷酸化調節(jié)作用及對其轉錄活性的影響。 方法: 用貼塊法分離培養(yǎng)大鼠主動脈內皮細胞(rat aortic endothelial cells,RAECs),倒置顯微鏡下觀察細胞形態(tài),并用免疫細胞化學法檢測內皮細胞表面Ⅷ因子相關抗原和活細胞攝取Dil-Ac-LDL的方法鑒定RAECs純度。用western blot法檢測RAECs和人血管內皮細胞株EA.hy926中FoxO各亞型FoxO1、FoxO3a和FoxO4的表達。亞融合狀態(tài)的血管內皮細胞經血清饑餓處理24小時后用于實驗。為檢測不同程度的氧化應激對FoxO1磷酸化的影響,分別以200μmol/L、500μmol/L或1000μmol/L H2O2處理血管內皮細胞20min,收集細胞總蛋白,用western blot法檢測FoxO1的S256位點和T24位點的磷酸化水平。選取500μmol/L濃度的H2O2刺激血管內皮細胞20min、30min或60min后,用western blot法檢測FoxO1的磷酸化與刺激時間的關系。為明確FoxO1的磷酸化是否與PI3K/Akt信號通路有關,我們檢測不同程度和不同作用時間的氧化應激對蛋白激酶Akt磷酸化的影響,同時運用兩種特異性的PI3K抑制劑LY294002或wortmannin預先阻斷該信號通路,用western blot法觀察氧化應激對FoxO1及Akt磷酸化水平的影響。為觀察氧化應激引起的磷酸化對FoxO1亞細胞定位的影響,在500μmol/L H2O2刺激RAECs20min后,我們分別提取了細胞漿與細胞核蛋白,用western blot法檢測磷酸化FoxO1的分布,并利用免疫細胞化學方法和熒光顯微鏡觀察磷酸化FoxO1的亞細胞定位。為進一步研究氧化應激誘導的FoxO1磷酸化對其轉錄活性的影響,將RAECs共轉染含有FoxO結合序列IRS(insulin-responsive sequence)的熒光素酶報告基因質粒(3×IRS-luc)和FoxO1的表達質粒或對照質粒, 24小時后給予細胞200μmol/L H2O2刺激12小時,裂解細胞,用雙熒光素酶報告基因試劑盒測定熒光素酶的活性。最后,我們檢測了氧化應激引起的FoxO1磷酸化對其靶基因Bim轉錄的影響。10μmol/L LY294002預處理RAECs 1h后給予200μmol/L H2O2刺激4h,提取細胞的總RNA,通過Real-time PCR方法檢測靶基因Bim的mRNA水平。 結果: 免疫細胞化學和活細胞攝取Dil-Ac-LDL的方法鑒定RAECs純度為90%以上。RAECs和EA.hy926細胞株內FoxO1及FoxO3a表達豐富。血管內皮細胞在不同濃度H2O2作用20min后,FoxO1的S256位點和T24位點出現(xiàn)磷酸化,其強度呈濃度依賴性;而500μmol/L作用血管內皮細胞后,FoxO1的S256位點和T24位點磷酸化在20min、30min及60min時都能檢測到。H2O2刺激對細胞中總FoxO1的含量沒有明顯影響。Akt的磷酸化呈現(xiàn)與FoxO1相似的濃度和時間依賴性。H2O2誘導的Akt及FoxO1的磷酸化可以被10μmol/L LY294002或500nmmol/L wortmannin預處理所抑制。500μmol/L H2O2作用RAECs20min后,細胞漿組分中磷酸化的FoxO1明顯增多,并可以觀察到細胞漿中磷酸化FoxO1(T24)熒光的增強。通過報告基因檢測發(fā)現(xiàn),FoxO1能明顯激活3×IRS-luc報告基因的熒光素酶活性,而H2O2對這一效應有明顯的抑制作用。Real-time PCR檢測結果顯示,Bim mRNA表達水平在磷酸化的影響,同時運用兩種特異性的PI3K抑制劑LY294002或wortmannin預先阻斷該信號通路,用western blot法觀察氧化應激對FoxO1及Akt磷酸化水平的影響。為觀察氧化應激引起的磷酸化對FoxO1亞細胞定位的影響,在500μmol/L H2O2刺激RAECs20min后,我們分別提取了細胞漿與細胞核蛋白,用western blot法檢測磷酸化FoxO1的分布,并利用免疫細胞化學方法和熒光顯微鏡觀察磷酸化FoxO1的亞細胞定位。為進一步研究氧化應激誘導的FoxO1磷酸化對其轉錄活性的影響,將RAECs共轉染含有FoxO結合序列IRS(insulin-responsive sequence)的熒光素酶報告基因質粒(3×IRS-luc)和FoxO1的表達質�；驅φ召|粒, 24小時后給予細胞200μmol/L H2O2刺激12小時,裂解細胞,用雙熒光素酶報告基因試劑盒測定熒光素酶的活性。最后,我們檢測了氧化應激引起的FoxO1磷酸化對其靶基因Bim轉錄的影響。10μmol/LY294002預處理RAECs 1h后給予200μmol/L H2O2刺激4h,提取細胞的總RNA,通過Real-time PCR方法檢測靶基因Bim的mRNA水平。H2O2刺激內皮細胞后顯著增高, PI3K抑制劑LY294002預處理則進一步增加了Bim的表達。 結論: 氧化應激引起血管內皮細胞中轉錄因子FoxO1的S256位點和T24位點磷酸化,其磷酸化水平呈一定的濃度與時間依賴性。上述位點的磷酸化是通過PI3K/Akt信號通路介導的。磷酸化導致FoxO1從細胞核轉移至細胞漿,并對其轉錄活性有負性調控作用。
[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.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R363

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