解偶联蛋白2在血管紧张素II致线粒体活性氧生成中的作用
发布时间：2018-08-23 08:46
【摘要】： 背景:大量的研究认为:活性氧类物(reactive oxygen species,ROS),如超氧阴离子和过氧化氢等)参与多种病理生理过程,包括内皮舒张功能减退,系统性高血压,细胞凋亡和炎症反应以及触发细胞内信号转导途径,参与许多疾病包括心血管疾病、神经变性、炎性疾病及感染;ROS在血管紧张素II(AngII)参与的病理生理活动中起重要的作用;解偶联蛋白2(Uncoupling protein-2,UCP2)属线粒体阴离子携带者一大家族的成员之一,研究表明UCP2可以通过使氧化磷酸化与ATP合成解偶联而降低线粒体内膜电势,从而抑制ROS的生成。 目的:观察血管紧张素II(AngII)对内皮细胞线粒体膜电位和活性氧的影响,探讨解偶联蛋白2(UCP2)在该途径中的作用 方法:实验共分三部分。(1)为了观察AngII对内皮细胞活性氧生成的影响,按干预浓度分为四组:对照组(DMEM培养基),AngII0.1μmol/L组,AngII1μmol/L组,AngII10μmol/L组分别与HUVEC(原代培养的人脐静脉内皮细胞)共同孵育24小时,RT-PCR法定量UCP2mRNA表达,DCFH-DA检测线粒体活性氧(流式细胞仪法),罗丹明123检测线粒体膜电位(流式细胞仪法); (2)为了观察UCP2对内皮细胞活性氧的影响,首先通过UCP2反义寡核苷酸降低UCP2表达,实验分组:对照组(DMEM培养基),UCP2反义寡核苷酸(浓度10μmol/L)组,UCP2错义寡核苷酸(浓度10μmol/L)组,然后用外源性解偶联剂增加内皮细胞解偶联活性,实验分组:对照组(DMEM培养基),AngII1μmol/L组,CLCCP(100μmol/L)+ AngII1μmol/L组,助溶剂DMSO100μmol/L+ AngII1μmol/L组,分别与HUVEC共培养24小时,DCFH-DA检测线粒体活性氧,罗丹明123检测线粒体膜电位。(3)观察氧化剂对UCP2的影响,实验分组:对照组(DMEM培养基),H2O2100μmol/L组,H2O250μmol/L组与HUVECs共培养24小时,RT-PCR法定量UCP2mRNA表达。 结果:用10μmol/L,1μmol/L,0.1μmol/L的AngII处理HUVEC24小时后1)流式细胞仪测线粒体膜电位(罗丹明123染色)的平均荧光强度分别为418.1±5.8,354.1±4.2,267.6±10.6(各组间比较p均0.01)。流式细胞仪测活性氧(DCFH-DA法)的平均荧光强度是365.3±5.2,258.2±4.8,188.6±12.6(各组间比较p均0.01),2)UCP2mRNA表达(灰度值)分别是0.450±0.024, 0.436±0.018, 0.381±0.020,1μmol/L组比0.1μmol/L组表达增加极显著(p0.01), 10μmol/L组比1μmol/L组表达增加显著(p0.05),即UCP2随AngII的浓度升高而表达增加;用UCP2反义寡核苷酸(浓度10微摩尔/升)与HUVEC共培养24小时后,与对照组相比,活性氧升高83%(p0.01),线粒体膜电位升高64%(p0.01), CLCCP(浓度100微摩尔/升)与HUVEC共培养24小时后,与对照组相比,活性氧下降26%(p0.01),线粒体膜电位下降28%(p0.01);此外用浓度为100微摩尔/升和50微摩尔/升的H2O2干预HUVECs24小时,UCP2mRNA表达为0.412±0.026和0.393±0.018,50μmol/L组比对照组表达增加极显著(p0.01), 100μmol/L组比50μmol/L组表达增加显著(p0.05),即UCP2随H2O2的浓度升高而表达增加。 结论:AngII增加线粒体活性氧的产生,上调UCP2的表达;降低UCP2的表达使活性氧生成增加,增加线粒体解偶联活性(CLCCP)使线粒体活性氧生成减少,提示AngII可能通过影响UCP2的表达从而影响线粒体活性氧的生成,也即UCP2具有抗氧化作用,通过提高解偶联蛋白2的表达从而减轻氧化应激可能是细胞抗氧化的重要机制。
[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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363
本文编号：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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363
【参考文献】
相关期刊论文 前1条
1 余继海,许戈良,汪建,荚卫东,傅斌生;人脐静脉内皮细胞的培养及鉴定[J];安徽医学;2003年05期
，本文编号：2198524
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