解偶聯(lián)蛋白2在血管緊張素II致線粒體活性氧生成中的作用
發(fā)布時(shí)間：2018-08-23 08:46
【摘要】： 背景:大量的研究認(rèn)為:活性氧類物(reactive oxygen species,ROS),如超氧陰離子和過氧化氫等)參與多種病理生理過程,包括內(nèi)皮舒張功能減退,系統(tǒng)性高血壓,細(xì)胞凋亡和炎癥反應(yīng)以及觸發(fā)細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)途徑,參與許多疾病包括心血管疾病、神經(jīng)變性、炎性疾病及感染;ROS在血管緊張素II(AngII)參與的病理生理活動(dòng)中起重要的作用;解偶聯(lián)蛋白2(Uncoupling protein-2,UCP2)屬線粒體陰離子攜帶者一大家族的成員之一,研究表明UCP2可以通過使氧化磷酸化與ATP合成解偶聯(lián)而降低線粒體內(nèi)膜電勢(shì),從而抑制ROS的生成。 目的:觀察血管緊張素II(AngII)對(duì)內(nèi)皮細(xì)胞線粒體膜電位和活性氧的影響,探討解偶聯(lián)蛋白2(UCP2)在該途徑中的作用 方法:實(shí)驗(yàn)共分三部分。(1)為了觀察AngII對(duì)內(nèi)皮細(xì)胞活性氧生成的影響,按干預(yù)濃度分為四組:對(duì)照組(DMEM培養(yǎng)基),AngII0.1μmol/L組,AngII1μmol/L組,AngII10μmol/L組分別與HUVEC(原代培養(yǎng)的人臍靜脈內(nèi)皮細(xì)胞)共同孵育24小時(shí),RT-PCR法定量UCP2mRNA表達(dá),DCFH-DA檢測(cè)線粒體活性氧(流式細(xì)胞儀法),羅丹明123檢測(cè)線粒體膜電位(流式細(xì)胞儀法); (2)為了觀察UCP2對(duì)內(nèi)皮細(xì)胞活性氧的影響,首先通過UCP2反義寡核苷酸降低UCP2表達(dá),實(shí)驗(yàn)分組:對(duì)照組(DMEM培養(yǎng)基),UCP2反義寡核苷酸(濃度10μmol/L)組,UCP2錯(cuò)義寡核苷酸(濃度10μmol/L)組,然后用外源性解偶聯(lián)劑增加內(nèi)皮細(xì)胞解偶聯(lián)活性,實(shí)驗(yàn)分組:對(duì)照組(DMEM培養(yǎng)基),AngII1μmol/L組,CLCCP(100μmol/L)+ AngII1μmol/L組,助溶劑DMSO100μmol/L+ AngII1μmol/L組,分別與HUVEC共培養(yǎng)24小時(shí),DCFH-DA檢測(cè)線粒體活性氧,羅丹明123檢測(cè)線粒體膜電位。(3)觀察氧化劑對(duì)UCP2的影響,實(shí)驗(yàn)分組:對(duì)照組(DMEM培養(yǎng)基),H2O2100μmol/L組,H2O250μmol/L組與HUVECs共培養(yǎng)24小時(shí),RT-PCR法定量UCP2mRNA表達(dá)。 結(jié)果:用10μmol/L,1μmol/L,0.1μmol/L的AngII處理HUVEC24小時(shí)后1)流式細(xì)胞儀測(cè)線粒體膜電位(羅丹明123染色)的平均熒光強(qiáng)度分別為418.1±5.8,354.1±4.2,267.6±10.6(各組間比較p均0.01)。流式細(xì)胞儀測(cè)活性氧(DCFH-DA法)的平均熒光強(qiáng)度是365.3±5.2,258.2±4.8,188.6±12.6(各組間比較p均0.01),2)UCP2mRNA表達(dá)(灰度值)分別是0.450±0.024, 0.436±0.018, 0.381±0.020,1μmol/L組比0.1μmol/L組表達(dá)增加極顯著(p0.01), 10μmol/L組比1μmol/L組表達(dá)增加顯著(p0.05),即UCP2隨AngII的濃度升高而表達(dá)增加;用UCP2反義寡核苷酸(濃度10微摩爾/升)與HUVEC共培養(yǎng)24小時(shí)后,與對(duì)照組相比,活性氧升高83%(p0.01),線粒體膜電位升高64%(p0.01), CLCCP(濃度100微摩爾/升)與HUVEC共培養(yǎng)24小時(shí)后,與對(duì)照組相比,活性氧下降26%(p0.01),線粒體膜電位下降28%(p0.01);此外用濃度為100微摩爾/升和50微摩爾/升的H2O2干預(yù)HUVECs24小時(shí),UCP2mRNA表達(dá)為0.412±0.026和0.393±0.018,50μmol/L組比對(duì)照組表達(dá)增加極顯著(p0.01), 100μmol/L組比50μmol/L組表達(dá)增加顯著(p0.05),即UCP2隨H2O2的濃度升高而表達(dá)增加。 結(jié)論:AngII增加線粒體活性氧的產(chǎn)生,上調(diào)UCP2的表達(dá);降低UCP2的表達(dá)使活性氧生成增加,增加線粒體解偶聯(lián)活性(CLCCP)使線粒體活性氧生成減少,提示AngII可能通過影響UCP2的表達(dá)從而影響線粒體活性氧的生成,也即UCP2具有抗氧化作用,通過提高解偶聯(lián)蛋白2的表達(dá)從而減輕氧化應(yīng)激可能是細(xì)胞抗氧化的重要機(jī)制。
[Abstract]:BACKGROUND: Many studies have shown that reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide, are involved in a variety of pathophysiological processes, including endothelial dysfunction, systemic hypertension, apoptosis and inflammation, as well as triggering intracellular signaling pathways, and are involved in many diseases including cardiovascular diseases. Neuropathy, inflammatory diseases and infections; ROS plays an important role in the pathophysiological activities of angiotensin II (AngII); Uncoupling protein-2 (UCP2) is a member of a large family of mitochondrial anion carriers. Studies have shown that UCP2 can reduce mitochondria by uncoupling oxidative phosphorylation and ATP synthesis. The membrane potential in vivo inhibits the generation of ROS.
AIM: To observe the effects of angiotensin II (AngII) on mitochondrial membrane potential and reactive oxygen species (ROS) in endothelial cells and to explore the role of uncoupling protein 2 (UCP2) in this pathway.
Methods: The experiment was divided into three parts. (1) To observe the effect of AngII on reactive oxygen species (ROS) production in human umbilical vein endothelial cells, four groups were divided according to the intervention concentration: control group (DMEM medium), AngII 0.1 micromol/L group, AngII 1 micromol/L group, AngII 10 micromol/L group and HUVEC (primary cultured human umbilical vein endothelial cells) incubated together for 24 hours, and the UCP2 mRNA table was quantified by RT-PCR. D, DCFH-DA detection of mitochondrial reactive oxygen species (flow cytometry), rhodamine 123 detection of mitochondrial membrane potential (flow cytometry); 2) in order to observe the effect of UCP2 on endothelial cells reactive oxygen species, first through UCP2 antisense oligonucleotides to reduce the expression of UCP2, experimental groups: control group (DMEM medium), UCP2 antisense oligonucleotides (concentration of 10 micromol/L) group, UCP2 missense oligonucleotide (concentration 10 micromol/L) group, then exogenous uncoupling agent was used to increase the uncoupling activity of endothelial cells. The experimental groups were divided into control group (DMEM medium), AngII1 micromol/L group, CLCCP (100 micromol/L) + AngII1 micromol/L group, cosolvent DMSO100 micromol/L + AngII1 micromol/L group, co-cultured with HUVEC for 24 hours, and mitochondrial activity was detected by DCFH-DA. Mitochondrial membrane potential was detected by sexual oxygen and Rhodamine 123. (3) The effects of oxidants on UCP2 were observed. The experimental groups were divided into control group (DMEM medium), H2O2100 micromol/L group, H2O250 micromol/L group and HUVECs co-cultured for 24 hours. The expression of UCP2 mRNA was quantified by RT-PCR.
Results: The average fluorescence intensity of mitochondrial membrane potential (rhodamine 123 staining) measured by flow cytometry was 418.1 (+ 5.8), 354.1 (+ 4.2) and 267.6 (+ 10.01) respectively after treating HUVEC with AngII at 10, 1 and 0.1 (+ 1) micromol / L for 24 hours. The average fluorescence intensity of reactive oxygen species (DCFH-DA) measured by flow cytometry was 365.3 (+ 5.2), 258.2 (+ 4.2 (+ 4.2). The expression of UCP2 mRNA (gray value) was 0.450 (+0.024), 0.436 (+0.018), 0.381 (+0.020) and 1-micromol/L groups, respectively. The expression of UCP2 increased significantly (p0.01) compared with 0.1-micromol/L group and 10-micromol/L group (p0.05), that is, the expression of UCP2 increased with the concentration of AngII. Compared with the control group, the reactive oxygen species (ROS) increased by 83% (p0.01), the mitochondrial membrane potential increased by 64% (p0.01), the reactive oxygen species (ROS) decreased by 26% (p0.01) and the mitochondrial membrane potential decreased by 28% (p0.01) after co-culture with HUVEC for 24 hours. The expression of UCP2 mRNA in HUVECs treated with L/L and 50 micromol/L H2O2 for 24 hours was significantly higher than that in control group (p0.01). The expression of UCP2 in 100 micromol/L group was significantly higher than that in 50 micromol/L group (p0.05).
Conclusion: AngII can increase the production of reactive oxygen species (ROS) in mitochondria and up-regulate the expression of UCP2, decrease the expression of UCP2 and increase the production of reactive oxygen species (ROS) in mitochondria, and increase the uncoupling activity (CLCCP) of mitochondria, suggesting that AngII may affect the production of reactive oxygen species (ROS) in mitochondria by affecting the expression of UCP2. Increasing the expression of uncoupling protein 2 and alleviating oxidative stress may be an important mechanism of cell antioxidant activity.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363
本文編號(hào)：2198524
[Abstract]:BACKGROUND: Many studies have shown that reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide, are involved in a variety of pathophysiological processes, including endothelial dysfunction, systemic hypertension, apoptosis and inflammation, as well as triggering intracellular signaling pathways, and are involved in many diseases including cardiovascular diseases. Neuropathy, inflammatory diseases and infections; ROS plays an important role in the pathophysiological activities of angiotensin II (AngII); Uncoupling protein-2 (UCP2) is a member of a large family of mitochondrial anion carriers. Studies have shown that UCP2 can reduce mitochondria by uncoupling oxidative phosphorylation and ATP synthesis. The membrane potential in vivo inhibits the generation of ROS.
AIM: To observe the effects of angiotensin II (AngII) on mitochondrial membrane potential and reactive oxygen species (ROS) in endothelial cells and to explore the role of uncoupling protein 2 (UCP2) in this pathway.
Methods: The experiment was divided into three parts. (1) To observe the effect of AngII on reactive oxygen species (ROS) production in human umbilical vein endothelial cells, four groups were divided according to the intervention concentration: control group (DMEM medium), AngII 0.1 micromol/L group, AngII 1 micromol/L group, AngII 10 micromol/L group and HUVEC (primary cultured human umbilical vein endothelial cells) incubated together for 24 hours, and the UCP2 mRNA table was quantified by RT-PCR. D, DCFH-DA detection of mitochondrial reactive oxygen species (flow cytometry), rhodamine 123 detection of mitochondrial membrane potential (flow cytometry); 2) in order to observe the effect of UCP2 on endothelial cells reactive oxygen species, first through UCP2 antisense oligonucleotides to reduce the expression of UCP2, experimental groups: control group (DMEM medium), UCP2 antisense oligonucleotides (concentration of 10 micromol/L) group, UCP2 missense oligonucleotide (concentration 10 micromol/L) group, then exogenous uncoupling agent was used to increase the uncoupling activity of endothelial cells. The experimental groups were divided into control group (DMEM medium), AngII1 micromol/L group, CLCCP (100 micromol/L) + AngII1 micromol/L group, cosolvent DMSO100 micromol/L + AngII1 micromol/L group, co-cultured with HUVEC for 24 hours, and mitochondrial activity was detected by DCFH-DA. Mitochondrial membrane potential was detected by sexual oxygen and Rhodamine 123. (3) The effects of oxidants on UCP2 were observed. The experimental groups were divided into control group (DMEM medium), H2O2100 micromol/L group, H2O250 micromol/L group and HUVECs co-cultured for 24 hours. The expression of UCP2 mRNA was quantified by RT-PCR.
Results: The average fluorescence intensity of mitochondrial membrane potential (rhodamine 123 staining) measured by flow cytometry was 418.1 (+ 5.8), 354.1 (+ 4.2) and 267.6 (+ 10.01) respectively after treating HUVEC with AngII at 10, 1 and 0.1 (+ 1) micromol / L for 24 hours. The average fluorescence intensity of reactive oxygen species (DCFH-DA) measured by flow cytometry was 365.3 (+ 5.2), 258.2 (+ 4.2 (+ 4.2). The expression of UCP2 mRNA (gray value) was 0.450 (+0.024), 0.436 (+0.018), 0.381 (+0.020) and 1-micromol/L groups, respectively. The expression of UCP2 increased significantly (p0.01) compared with 0.1-micromol/L group and 10-micromol/L group (p0.05), that is, the expression of UCP2 increased with the concentration of AngII. Compared with the control group, the reactive oxygen species (ROS) increased by 83% (p0.01), the mitochondrial membrane potential increased by 64% (p0.01), the reactive oxygen species (ROS) decreased by 26% (p0.01) and the mitochondrial membrane potential decreased by 28% (p0.01) after co-culture with HUVEC for 24 hours. The expression of UCP2 mRNA in HUVECs treated with L/L and 50 micromol/L H2O2 for 24 hours was significantly higher than that in control group (p0.01). The expression of UCP2 in 100 micromol/L group was significantly higher than that in 50 micromol/L group (p0.05).
Conclusion: AngII can increase the production of reactive oxygen species (ROS) in mitochondria and up-regulate the expression of UCP2, decrease the expression of UCP2 and increase the production of reactive oxygen species (ROS) in mitochondria, and increase the uncoupling activity (CLCCP) of mitochondria, suggesting that AngII may affect the production of reactive oxygen species (ROS) in mitochondria by affecting the expression of UCP2. Increasing the expression of uncoupling protein 2 and alleviating oxidative stress may be an important mechanism of cell antioxidant activity.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 余繼海,許戈良,汪建,莢衛(wèi)東,傅斌生;人臍靜脈內(nèi)皮細(xì)胞的培養(yǎng)及鑒定[J];安徽醫(yī)學(xué);2003年05期
，本文編號(hào)：2198524
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