本文關(guān)鍵詞：氧化應(yīng)激對血管內(nèi)皮細(xì)胞中轉(zhuǎn)錄因子FoxO1磷酸化的調(diào)節(jié)作用　出處：《南京醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 過氧化氫 FoxO1 血管內(nèi)皮細(xì)胞 Akt

【摘要】： 目的: 內(nèi)皮功能障礙是許多心血管疾病的共同的危險因素。氧化應(yīng)激引起的細(xì)胞損傷是內(nèi)皮細(xì)胞功能障礙的重要機(jī)制之一。FoxO(Forkhead box O)是Forkhead轉(zhuǎn)錄因子的一個亞家族,它是一類重要的氧化還原敏感的轉(zhuǎn)錄因子,參與細(xì)胞周期調(diào)控、凋亡、氧化應(yīng)激抵抗等生物學(xué)過程。本文采用過氧化氫(H2O2)刺激血管內(nèi)皮細(xì)胞模擬氧化應(yīng)激模型,探討H2O2對細(xì)胞內(nèi)轉(zhuǎn)錄因子FoxO1的磷酸化調(diào)節(jié)作用及對其轉(zhuǎn)錄活性的影響。 方法: 用貼塊法分離培養(yǎng)大鼠主動脈內(nèi)皮細(xì)胞(rat aortic endothelial cells,RAECs),倒置顯微鏡下觀察細(xì)胞形態(tài),并用免疫細(xì)胞化學(xué)法檢測內(nèi)皮細(xì)胞表面Ⅷ因子相關(guān)抗原和活細(xì)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(nèi)皮細(xì)胞株EA.hy926中FoxO各亞型FoxO1、FoxO3a和FoxO4的表達(dá)。亞融合狀態(tài)的血管內(nèi)皮細(xì)胞經(jīng)血清饑餓處理24小時后用于實(shí)驗(yàn)。為檢測不同程度的氧化應(yīng)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(nèi)皮細(xì)胞20min,收集細(xì)胞總蛋白,用western blot法檢測FoxO1的S256位點(diǎn)和T24位點(diǎn)的磷酸化水平。選取500μmol/L濃度的H2O2刺激血管內(nèi)皮細(xì)胞20min、30min或60min后,用western blot法檢測FoxO1的磷酸化與刺激時間的關(guān)系。為明確FoxO1的磷酸化是否與PI3K/Akt信號通路有關(guān),我們檢測不同程度和不同作用時間的氧化應(yīng)激對蛋白激酶Akt磷酸化的影響,同時運(yùn)用兩種特異性的PI3K抑制劑LY294002或wortmannin預(yù)先阻斷該信號通路,用western blot法觀察氧化應(yīng)激對FoxO1及Akt磷酸化水平的影響。為觀察氧化應(yīng)激引起的磷酸化對FoxO1亞細(xì)胞定位的影響,在500μmol/L H2O2刺激RAECs20min后,我們分別提取了細(xì)胞漿與細(xì)胞核蛋白,用western blot法檢測磷酸化FoxO1的分布,并利用免疫細(xì)胞化學(xué)方法和熒光顯微鏡觀察磷酸化FoxO1的亞細(xì)胞定位。為進(jìn)一步研究氧化應(yīng)激誘導(dǎo)的FoxO1磷酸化對其轉(zhuǎn)錄活性的影響,將RAECs共轉(zhuǎn)染含有FoxO結(jié)合序列IRS(insulin-responsive sequence)的熒光素酶報告基因質(zhì)粒(3×IRS-luc)和FoxO1的表達(dá)質(zhì)�；�?qū)φ召|(zhì)粒, 24小時后給予細(xì)胞200μmol/L H2O2刺激12小時,裂解細(xì)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(yīng)激引起的FoxO1磷酸化對其靶基因Bim轉(zhuǎn)錄的影響。10μmol/L LY294002預(yù)處理RAECs 1h后給予200μmol/L H2O2刺激4h,提取細(xì)胞的總RNA,通過Real-time PCR方法檢測靶基因Bim的mRNA水平。 結(jié)果: 免疫細(xì)胞化學(xué)和活細(xì)胞攝取Dil-Ac-LDL的方法鑒定RAECs純度為90%以上。RAECs和EA.hy926細(xì)胞株內(nèi)FoxO1及FoxO3a表達(dá)豐富。血管內(nèi)皮細(xì)胞在不同濃度H2O2作用20min后,FoxO1的S256位點(diǎn)和T24位點(diǎn)出現(xiàn)磷酸化,其強(qiáng)度呈濃度依賴性;而500μmol/L作用血管內(nèi)皮細(xì)胞后,FoxO1的S256位點(diǎn)和T24位點(diǎn)磷酸化在20min、30min及60min時都能檢測到。H2O2刺激對細(xì)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(dǎo)的Akt及FoxO1的磷酸化可以被10μmol/L LY294002或500nmmol/L wortmannin預(yù)處理所抑制。500μmol/L H2O2作用RAECs20min后,細(xì)胞漿組分中磷酸化的FoxO1明顯增多,并可以觀察到細(xì)胞漿中磷酸化FoxO1(T24)熒光的增強(qiáng)。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(yīng)有明顯的抑制作用。Real-time PCR檢測結(jié)果顯示,Bim mRNA表達(dá)水平在磷酸化的影響,同時運(yùn)用兩種特異性的PI3K抑制劑LY294002或wortmannin預(yù)先阻斷該信號通路,用western blot法觀察氧化應(yīng)激對FoxO1及Akt磷酸化水平的影響。為觀察氧化應(yīng)激引起的磷酸化對FoxO1亞細(xì)胞定位的影響,在500μmol/L H2O2刺激RAECs20min后,我們分別提取了細(xì)胞漿與細(xì)胞核蛋白,用western blot法檢測磷酸化FoxO1的分布,并利用免疫細(xì)胞化學(xué)方法和熒光顯微鏡觀察磷酸化FoxO1的亞細(xì)胞定位。為進(jìn)一步研究氧化應(yīng)激誘導(dǎo)的FoxO1磷酸化對其轉(zhuǎn)錄活性的影響,將RAECs共轉(zhuǎn)染含有FoxO結(jié)合序列IRS(insulin-responsive sequence)的熒光素酶報告基因質(zhì)粒(3×IRS-luc)和FoxO1的表達(dá)質(zhì)�；�?qū)φ召|(zhì)粒, 24小時后給予細(xì)胞200μmol/L H2O2刺激12小時,裂解細(xì)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(yīng)激引起的FoxO1磷酸化對其靶基因Bim轉(zhuǎn)錄的影響。10μmol/LY294002預(yù)處理RAECs 1h后給予200μmol/L H2O2刺激4h,提取細(xì)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(nèi)皮細(xì)胞后顯著增高, PI3K抑制劑LY294002預(yù)處理則進(jìn)一步增加了Bim的表達(dá)。 結(jié)論: 氧化應(yīng)激引起血管內(nèi)皮細(xì)胞中轉(zhuǎn)錄因子FoxO1的S256位點(diǎn)和T24位點(diǎn)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(diǎn)的磷酸化是通過PI3K/Akt信號通路介導(dǎo)的。磷酸化導(dǎo)致FoxO1從細(xì)胞核轉(zhuǎn)移至細(xì)胞漿,并對其轉(zhuǎn)錄活性有負(fù)性調(diào)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(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R363

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 于竹芹;于雪蓮;李生堯;王蘇;段德麟;;羊棲菜對四氧嘧啶糖尿病模型小鼠血糖水平影響[J];青島大學(xué)醫(yī)學(xué)院學(xué)報;2011年01期

2 馬璇;劉琳;張蕊;劉吉東;沈衛(wèi);;藻藍(lán)蛋白對2型糖尿病大鼠胰腺組織iNOS表達(dá)的影響[J];當(dāng)代醫(yī)學(xué);2010年07期

3 肖勝;陳婧;汪宏良;肖勁松;;癲癇持續(xù)狀態(tài)患者神經(jīng)元損傷與氧化應(yīng)激的關(guān)系[J];國際檢驗(yàn)醫(yī)學(xué)雜志;2012年06期

4 王家澤;張繼紅;王霞;;Quercetin調(diào)節(jié)肝癌HepG2細(xì)胞PTEN基因表達(dá)誘導(dǎo)細(xì)胞凋亡的實(shí)驗(yàn)研究[J];嶺南現(xiàn)代臨床外科;2008年05期

5 馬麗;陳旭;;失巢凋亡與腫瘤關(guān)系的研究進(jìn)展[J];山東醫(yī)藥;2012年12期

6 謝飛舟;施冬云;肖玲;劉精東;劉珊林;;2型糖尿病葡萄糖應(yīng)激與抗氧化代償?shù)淖兓痆J];復(fù)旦學(xué)報(醫(yī)學(xué)版);2009年01期

7 謝飛舟;施冬云;肖玲;劉珊林;;糖應(yīng)激對2型糖尿病氧化還原代謝水平的影響[J];生物物理學(xué)報;2009年S1期

8 張琨;岳宗相;謝春光;;中醫(yī)藥抗氧化治療糖尿病血管并發(fā)癥的研究概況[J];時珍國醫(yī)國藥;2011年09期

9 吳琴;左芳;尹義軍;;癲癇全面強(qiáng)直-陣攣發(fā)作患者的氧化應(yīng)激水平[J];微循環(huán)學(xué)雜志;2011年04期

10 朱婉兒;胡長春;謝文婷;吳麗霞;陳芝蕓;梅垣宏行;;慢性應(yīng)激對大鼠海馬Akt/FKHRL1信號通路活性的影響[J];心理學(xué)報;2007年04期

相關(guān)會議論文 前1條

1 謝飛舟;施冬云;肖玲;劉珊林;;糖應(yīng)激對2型糖尿病氧化還原代謝水平的影響[A];第十一次中國生物物理學(xué)術(shù)大會暨第九屆全國會員代表大會摘要集[C];2009年

相關(guān)博士學(xué)位論文 前10條

1 丁威;二花臉豬卵泡形成和早期發(fā)育的特性及其相關(guān)通路的研究[D];南京農(nóng)業(yè)大學(xué);2010年

2 祁永福;疏肝和胃降逆顆粒對反流性食管炎大鼠的治療作用及其機(jī)制研究[D];蘭州大學(xué);2011年

3 李學(xué)斌;基因效應(yīng)與信號轉(zhuǎn)導(dǎo)數(shù)學(xué)模型的建立及應(yīng)用[D];南京農(nóng)業(yè)大學(xué);2006年

4 張雄飛;地塞米松部分通過Foxol轉(zhuǎn)錄因子抑制胰島β細(xì)胞內(nèi)PDX-1因子[D];南京醫(yī)科大學(xué);2007年

5 王永忠;內(nèi)源性一氧化氮對胃癌細(xì)胞凋亡的影響及信號轉(zhuǎn)導(dǎo)機(jī)制研究[D];南京醫(yī)科大學(xué);2007年

6 龐衛(wèi)軍;豬FoxO1基因cDNA的克隆及對前體脂肪細(xì)胞和成肌細(xì)胞分化的調(diào)控作用[D];西北農(nóng)林科技大學(xué);2007年

7 孟春花;香豬卵巢細(xì)胞生物學(xué)特性和影響母豬卵巢顆粒細(xì)胞體外成熟的因素探討[D];南京農(nóng)業(yè)大學(xué);2008年

8 陳昊;FOXO3a基因在Anoikis中對細(xì)胞的保護(hù)作用研究[D];浙江大學(xué);2009年

9 王玲;普通牛FoxO1、FoxO3、FoxO4基因的克隆、表達(dá)及其對肉質(zhì)性狀的遺傳效應(yīng)分析[D];四川農(nóng)業(yè)大學(xué);2010年

10 易波;ROS/AMPK介導(dǎo)的凋亡通路在川芎嗪抗胃癌作用中的機(jī)制研究[D];南昌大學(xué)醫(yī)學(xué)院;2013年

相關(guān)碩士學(xué)位論文 前10條

1 崔久彩;急性應(yīng)激對海馬Akt/FKHRL1信號通路活性改變的影響[D];浙江大學(xué);2011年

2 王芳;SMND-309的體外抗氧化活性研究[D];陜西師范大學(xué);2011年

3 周國紅;短期胰島素強(qiáng)化治療對新診斷2型糖尿病患者IκB-α、COX-2、iNOS表達(dá)的影響[D];河北醫(yī)科大學(xué);2011年

4 王蕾蕾;葡萄糖負(fù)荷對血管內(nèi)皮功能和氧化應(yīng)激的影響及那格列奈的干預(yù)作用[D];北京協(xié)和醫(yī)學(xué)院;2011年

5 陳步遠(yuǎn);黃芩苷對HL-60細(xì)胞增殖、凋亡、分化的影響及其作用機(jī)制的探討[D];福建醫(yī)科大學(xué);2006年

6 韓敏娜;糖尿病微血管病變患者血小板參數(shù)及微量元素鋅的變化及其臨床意義[D];吉林大學(xué);2009年

7 朱玲;PI3K在ALS萎縮肌纖維中表達(dá)的意義[D];河北醫(yī)科大學(xué);2009年

8 楊曉寧;三葉因子1對誘導(dǎo)性的ICAM1與VCAM1表達(dá)的影響[D];廈門大學(xué);2009年

9 尉希清;辛伐他汀對高糖誘導(dǎo)乳鼠心肌細(xì)胞損傷的干預(yù)作用及其機(jī)制研究[D];蘇州大學(xué);2010年

10 張磊;異丙腎上腺素通過NF-kappaB通路延緩失神經(jīng)骨骼肌萎縮[D];山西醫(yī)科大學(xué);2010年

，

本文編號：1357389