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Objective To investigate the changes in reactive oxygen species (ROS) and dimethylarginine dimethylaminohydrolase-asymmetric dimethylarginine (DDAH-ADMA) system in the process of endothelial cell senescence after exposure to high glucose. Methods The human umbilical vein endothelial cells (HUVECs) were cultured with different concentrations of glucose, e.g. 5. 5 mmol/L (normal level),and high levels as 11. 0, 22. 0 and 33. 0 mmol/L. for 48 hours, respectively. Subsequently, SA-β-gal staining was used to evaluate senescence of cells. Telomerase activity was detected by polymerase chain reactionenzyme linked immunosorbent assay (PCR-ELISA). The intracellular ROS level was measured by flow cytometry. The ADMA concentration and DDAH activity were determined with high-performance liquid chromatography. Results Compared with normal glucose concentration group, after the endothelial cells were treated with high glucose concentration (11. 0 - 33. 0 mmol/L) for 48 hours, the number of SA-β-gal positive cells was increased significantly [(7.00±1. 73)%, (12. 67±2. 03)%, (16. 00±2. 26)% vs. (4. 00±1.33)%, P>0.05, P<0.05, P<0.05] and the telomerase activity was inhibited dramatically [(91. 32±4.01)%, (78. 44±3. 78)%, (56. 04±3. 35)% vs. 100%, all P<0. 05]. The ROS level (mfi) was increased in all high glucose groups (159. 84±27. 52, 188. 99±18. 77, 244. 56±20. 96 vs. 117.11±18. 76, P<0. 05 or P<0. 01). At the same time, the ADMA (μmol/L) production was increased (0. 78±0. 14, 0. 88±0.18,1. 08±0.15 vs. 0. 70±0. 12, P>0. 05, P<0. 05, P<0. 05), and DDAH activity was decreased [(91. 32±4.01)%, (78.44±3.78)%, (56. 04± 3. 35)% vs. 100%, all P<0.05]. Conclusion High glucose can accelerate endothelial cells senescence in dose-dependent manner and the underlying mechanism may be related to an increased oxidative stress and change in DDAH-ADMA system. 相似文献
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高糖诱导人脐静脉内皮细胞衰老过程中活性氧和一氧化氮合酶/一氧化氮系统的变化 总被引:2,自引:0,他引:2
目的 观察高糖加速内皮细胞衰老过程中细胞内活性氧(ROS)水平和一氧化氮合酶/一氧化氮(NOS/NO)系统的变化.方法 将正常浓度糖(5.5 mmol/L)和高浓度糖(分别为11、22、33 mmol/L)培养液作用于人脐静脉内皮细胞48 h后,用β-半乳糖苷酶(β-gal)染色鉴定衰老细胞,用聚合酶链反应-酶联免疫吸附法(PCR-ELISA)检测端粒酶活性,用硝酸盐还原酶法检测细胞上清液总NO水平,用蛋白质免疫印迹法(Western blotting)分析内皮型一氧化氮合酶(eNOS)蛋白表达,用荧光酶标仪检测细胞内NOS活性,用流式细胞仪检测细胞内ROS水平.结果 高糖培养液作用于内皮细胞48 h后,与对照组比较,随糖浓度升高内皮细胞β-gal染色阳性细胞数明显增多,端粒酶活性下降,细胞NO合成减少,NOS活性被抑制,细胞内ROS水平明显升高(P<0.05或P<0.01),但eNOS蛋白表达变化不明显.结论 高糖加速内皮细胞衰老进程,其作用机制可能与增强氧化应激,抑制NOS/NO系统有关. 相似文献
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目的研究血液透析(Hemodialysis,HD)患者血浆非对称性二甲基精氨酸(Asymmetric dimethylarginine,ADMA)与透析中血压变化的关系。方法经生物电阻抗检测干体质量达标且符合入选标准的维持性血液透析(Maintenance Hemodialysis,MHD)患者31名进入研究,根据血液透析过程中血压波动情况分为年龄相匹配的3组:透析中高血压组(n=11)、低血压组(n=12)和血压平稳组(n=8)。用酶联免疫吸附(Enzyme linked immunosorbent assay,ELISA)法检测患者透析前、后血浆ADMA水平,探讨ADMA与透析中血压变化的关系,并进行组间矿物质骨代谢指标、电解质、营养指标、炎性标记物、血脂水平、脉压差和降压治疗等的比较。结果 31例MHD患者透析前血ADMA均值为3.37±1.48μmol/L,透析后降至1.71±0.80μmol/L(P0.001),均显著高于国外正常参考值。透析中低血压组透析前、后血ADMA值(4.38±1.56μmol/L,2.25±0.83μmol/L)均高于透析中高血压组和血压平稳组,差异有统计学意义(2.70±1.18μmol/L,1.32±0.60μmol/L和2.78±0.88μmol/L,1.43±0.56μmol/L;P=0.006和0.006)。透析中高血压组患者透析中的平均脉压差高于透析中低血压组和血压平稳组(62.41±11.57mmHg,48.80±12.88 mmHg和44.56±8.30 mmHg,P=0.004)。高血压组碱性磷酸酶(ALP)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)和高敏C-反应蛋白(high-sensitivity c-reactive protein,Hs-CRP)均高于血压平稳组(P值分别为0.036、0.039、0.046、0.046),低血压组同样指标也高于血压平稳组(P值分别为0.046、0.035、0.040、0.004),上述指标在高血压组和低血压组间差异无统计学意义(P0.05)。结论在干体质量达标的MHD患者中,血ADMA水平显著高于正常,透析过程中的血压波动与内皮功能不良、血管僵硬、微炎症状态等密切相关。 相似文献
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非对称性二甲基精氨酸水平与缺血性脑卒中的相关性 总被引:1,自引:0,他引:1
目的 探讨非对称性二甲基精氨酸(asymmetricaldimethylarginine,ADMA)对缺血性脑卒中诊断及病情判断的临床价值.方法 选取88例缺血性脑卒中患者,采用欧洲卒中量表(ESS)进行神经功能缺损评分分为轻度卒中组(31例)、中度卒中组(39例)和重度卒中组(18例).另选30名年龄、性别构成比与患者组差异无统计学意义的健康志愿者为对照组,分别用高效液相色谱联合质谱法测定血浆中ADMA的水平,分析血浆ADMA水平在对照组和缺血性脑卒中各亚组之间的相关性.结果 对照组、轻度卒中组、中度卒中组和重度卒中组的血浆ADMA水平(±s,μmol/L)分别为:1.0±0.10,2.90±0.30,4.82±0.21及6.61±0.23.与对照组血浆ADMA水平比较,患者组各亚组ADMA水平明显升高,其差异有统计学意义(P<0.05);轻度卒中组与中度和重度卒中组的差异有统计学意义(P<0.05);重度组与中度卒中组的差异有统计学意义(P<0.05),与轻度卒中组的差异有统计学显著性意义(P<0.001).结论 血浆ADMA水平与缺血性脑卒中的发病及病变严重程度相关,检测血浆ADMA水平的变化,对缺血性脑卒中的诊断和病情判断、指导治疗有一定的帮助. 相似文献
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目的:观察辛伐他汀对非对称性二甲基精氨酸(ADMA)引起的人脐静脉内皮细胞(HUVECs)炎症反应的抑制作用,探讨他汀类药物除降脂作用以外的其他抗动脉粥样硬化机制.方法:体外培养的人济静脉内皮细胞,待细胞生长到融合状态时加入不同浓度的ADMA(3、10、30μmol/L)作用48h,观察ADMA对内皮细胞炎症因子产生的影响.不同浓度的辛伐他汀预先孵育20min,然后加入ADMA(30 μmol/L)作用48h,用ELISA法检测上清液中MCP-1、IL-6和TNF-a浓度,比色法检测一氧化氮(NO)含量的变化.结果:ADMA呈剂量依赖性增加上清液中MCP-1、IL-6和TNF-α浓度并降低NO的含量.外源性补充辛伐他汀可逆转ADMA的效应,且呈剂量依赖性(P<0.05).用一氧化氮合酶抑制剂左旋硝基精氨酸甲酯(L-NAME)抑制一氧化氮合酶降低NO的合成后,可部分取消辛伐他汀的保护作用.结论:ADMA通过诱导炎症反应,引起内皮功能紊乱,其紊乱程度与ADMA的浓度有关;而辛伐他汀能以剂量依赖的方式抑制ADMA诱导的炎症反应,其机制除与其增加NO的合成有关外,可能还有其他物质或途径的参与. 相似文献
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目的:探讨非对称性二甲基精氨酸(ADMA)对内皮生长晕细胞(EOCs)凋亡和功能的影响.方法:密度梯度离心法分离脐血单个核细胞,培养并扩增EOCs,免疫组化法、荧光染色法鉴定其内皮细胞特性.将浓度为0、1、5、10、30μmol/L的ADMA与EOCs作用48 h,流式细胞仪检测细胞凋亡率,DAPI染色观察凋亡细胞核形态变化,并在倒置显微镜下检测细胞的黏附能力和成血管能力.结果:ADMA(1~30μmol/L)呈浓度依赖性的诱导EOCs的凋亡(P<0.01).ADMA处理组于DAPI染色下可见更显著的细胞凋亡形态学改变.除1μmol/L ADMA外,5、10、30 μmol/L ADMA均可抑制EOCs的黏附能力.10 μmol/L ADMA作用组与对照组相比,明显降低细胞成血管能力(P<0.01).结论:ADMA可以诱导体外培养的EOCs发生凋亡并抑制其黏附能力和成血管能力. 相似文献
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<正>非对称性二甲基精氨酸(ADMA)是精氨酸甲基化的衍生物,广泛分布于人体的组织细胞及体液中。ADMA是内源性一氧化氮合酶(e NOS)主要的抑制剂,而e NOS为合成一氧化氮(NO)所必需[1]。研究表明,血浆ADMA浓度升高会使NO的合成受抑,导致血管内皮功能障碍和动脉粥样硬化。1非对称性二甲基精氨酸的生成与代谢ADMA是含甲基化精氨酸残基的蛋白质,由特 相似文献
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背景:他汀类药物对血管内皮细胞的凋亡是否有影响目前尚不明确。目的:探讨辛伐他汀对高糖诱导的人脐静脉内皮细胞凋亡的影响。方法:用DMEM细胞培养液培养人脐静脉内皮细胞,将细胞分成空白对照组、高糖组和高糖+辛伐他汀组,用四甲基偶氮唑蓝比色法测定人脐静脉内皮细胞的存活率,流式细胞仪和Westernblot分别检测细胞早期凋亡率及P53蛋白表达。结果与结论:高糖组及高糖+辛伐他汀组细胞增殖率较空白对照组明显降低(P〈0.01),而高糖组细胞增殖率较高糖+辛伐他汀组亦降低(P〈0.01);高糖组P53蛋白表达量及凋亡率较空白对照组及高糖+辛伐他汀组明显增加(P〈0.01),高糖+辛伐他汀组P53蛋白表达及凋亡率亦明显高于空白对照组(P〈0.01)。表明高糖可通过促进促凋亡蛋白P53的表达进而促进人脐静脉内皮细胞的凋亡,而辛伐他汀可抑制此作用。 相似文献
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脑小血管病是一组不同病因引起脑小血管异常所致的脑组织局部病变.脑低灌注、血脑屏障通透性增加和血管内皮细胞功能障碍在脑小血管病的病理机制中发挥重要作用.血管内皮细胞功能障碍在脑小血管病病理机制中的作用目前尚未完全明确.非对称性二甲基精氨酸可减少血管活性物质一氧化氮的生成,导致血管内皮细胞功能障碍.非对称性二甲基精氨酸可通... 相似文献
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Erythropoietin (EPO) can induce a series of cytoprotective effects in many non-hematopoietic tissues through interaction with the erythropoietin receptor (EPOR), but whether EPO can prevent the overproduction of reactive oxygen species (ROS) and apoptosis in diabetes remains unclear. Here, we report that renal tubular cells possess EPOR and that EPO reduces high glucose-induced oxidative stress in renal tubular cells. Further, we found that EPO inhibited high glucose-induced renal tubular cell apoptosis and that this protective effect was dependent on reduction of Bax/caspase-3 expression as well as elevation of Bcl-2 expression. Our results suggest that EPO can inhibit high glucose-induced renal tubular cell apoptosis through direct effect on anti-oxidative stress and that EPOR may play a key role in this process. 相似文献
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三种抗癌剂诱导肿瘤细胞凋亡与活性氧产生关系的研究 总被引:3,自引:0,他引:3
目的 探讨抗癌剂、活性氧、凋亡三者之间的关系,从而进一步探讨抗癌剂的作用及肿瘤细胞耐药机制以及活性氧在肿瘤发生上的意义。方法 用足叶乙甙(Vp16)、阿霉素(ADR)、顺铂(DDP0作用于K562细胞,用流式细胞仪检测三种抗癌剂在不同浓度、作用24h对K562细胞活性氧产生的影响及对凋亡的影响。同时用形态学方法观察凋亡细胞的形态改变。结果 ①Vp16、ADR、DDP三种抗癌剂均可通过激发K562细胞产生活性氧来诱导该细胞发生凋亡;②每种抗癌剂激发K562细胞产生活性氧及诱发该细胞凋亡各有其最适浓度(Vp16为5μg/ml,ADR为3μg/ml,DDP为μg/ml)及最佳作用时间(24h)。结论 流式细胞仪(FCM)测定细胞内活性氧产生状态及细胞凋亡情况,方法具有敏感、快速、简便、只需微量血、可除去坏死细胞碎片等优点。可用于抗癌剂的筛选,指导临床用药及选择敏感抗癌剂的有效剂量及最佳时间,并为探索治疗肿瘤新方法提供一个思路。 相似文献
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Reactive oxygen species can be important mediators of damage to cell molecules and structures. Besides the endogen antioxidant defences, the antioxidant intake in the diet has an important role in the protection against the development of diseases produced by oxidative damage. Resveratrol is a naturally occurring compound present in many plants some of which are part of the human diet. This molecule has been thoroughly investigated because of its antioxidant and anticarcinogenic properties among others. We investigated whether resveratrol could provide protective antioxidant action in primary rat hepatocyte cultures. Primary rat hepatocytes cultures were exposed to 300 μM tert-butyl hydroperoxide; 25, 50 or 75 μM resveratrol or to 300 μM tert-butyl hydroperoxide plus 25, 50 or 75 μM resveratrol for different time periods. Necrosis was evaluated by lactate dehydrogenase liberation to the medium. Apoptosis was evaluated by caspase 3 activity measurement. Changes in cellular morphology after the different treatments were recorded using bright field microscopy. Inhibition of the reactive oxygen species by resveratrol was studied by confocal microscopy and spectrofluorimetrically. Resveratrol inhibited necrosis induced by tert-butyl hydroperoxide. No apoptosis was observed in any treatment. It also was effective in eliminating reactive oxygen species. At 75 μM, the highest concentration tested, resveratrol became slightly cytotoxic. Our results show that resveratrol protects primary rat hepatocytes in culture from oxidative stress induced cell death. These results suggest that resveratrol could enhance the antioxidant status of hepatic cells. 相似文献
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目的 探讨高糖对脂多糖(LPS)刺激下血管内皮细胞损伤的影响及其机制.方法 将人肺脏微血管内皮细胞(PMVEC)分为正常糖组(NG组)、正常糖+LPS刺激组(NGL组)、高糖组(HG组)、高糖+LPS刺激组(HGL组),分别给予含10%小牛血清的正常糖(5.5 mmol/L)或高糖(33 mmol/L)培养5d,加入10 mg/L LPS刺激细胞24h.采用免疫荧光染色观察细胞纤维肌动蛋白(F-actin)的分布及变化;扫描电镜观察细胞膜窗孔数量及孔径变化;细胞迁移实验(Transwell)测定单层内皮细胞的辣根过氧化物酶(HRP)通透性;硝酸盐还原法(Griess法)检测细胞培养上清液中一氧化氮(NO)含量;蛋白质免疫印迹试验(Western blotting)测定细胞二甲基精氨酸-二甲胺水解酶2(DDAH2)、诱生型一氧化氮合酶(iNOS)、内皮型一氧化氮合酶(eNOS)的蛋白表达.结果 与NGL组比较,HGL组PMVEC的F-actin分布排列紊乱,细胞膜窗孔异常增大、增多,细胞单层对HRP通透率增加[(53.62±6.70)%比(23.63±3.92)%,P<0.01],细胞DDAH2表达(积分A值)减少(0.33±0.08比0.77±0.14,P<0.01),iNOS表达(积分A值)增加(1.40±0.29比1.04±0.09,P<0.01),eNOS表达(积分A值)减少(0.67±0.09比0.91±0.17,P<0.05),上清液NO含量(μmol/L)增多(20.36±2.25比7.99±0.33,P<0.01).结论 高糖加重LPS刺激下体外培养PMVEC的F-actin分布紊乱、单层细胞通透性增加;NO调节紊乱可能参与了PMVEC损害的发生. 相似文献
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铁过载诱导活性氧物质生成对骨髓造血功能影响的体外实验研究 总被引:1,自引:0,他引:1
目的 建立体外铁过载骨髓造血细胞模型,检验铁过载对细胞活性氧物质(ROS)水平的影响以及ROS升高对骨髓造血功能的影响。方法 在骨髓单个核细胞培养的过程中添加枸橼酸铁铵(FAC),使细胞铁过载,检验这一过程中细胞ROS水平、细胞凋亡水平、造血细胞集落形成和CD 34+细胞计数的变化。再用去铁胺(DFO)祛铁或抗氧化剂N-乙酰半胱氨酸(NAC)清除过多的ROS后,检测上述指标的变化。结果 ①在培养液中加入不同浓度FAC培养不同时间,发现骨髓造血细胞内可变铁池(LIP)水平升高,且具有时间和浓度依赖性,在含400μmoL/L FAC的培养液中培养24h时LIP水平达到最高。②在400μmol/L FAC浓度下,培养骨髓造血细胞24h后骨髓造血细胞内总的ROS、粒细胞和红细胞内ROS显著升高,分别为对照组的1.77、1.75和2.12倍。与对照组比较,DFO和NAC处理后均能明显降低细胞内ROS水平(P<0.05)。③对骨髓细胞造血功能的检测发现FAC组细胞凋亡比例[(24.80±2.99)%]较对照组[(8.90±0.96)%]显著升高;造血细胞集落形成单位(CFUE、CFU-GM、BFU-E和CFU-mix)计数明显低于对照组(P值均<0.05);CD34+细胞比例[(0.39±0.07)%]较对照组[(0.91±0.12)%]也显著降低。且这些损伤都可以通过DFO和NAC处理而部分恢复。结论 铁过载通过诱导ROS生成影响骨髓造血功能,这种损伤可以通过祛铁和抗氧化处理减轻。可能为治疗铁过载患者骨髓造血功能低下寻找新的靶点。 相似文献
16.
目的探讨低浓度活性氧对肾癌786-O细胞先天性多药耐药的逆转作用及相关机制。方法采用WST-1细胞增殖及细胞毒性检测试剂盒确定活性氧H2O2的非细胞毒性剂量,以及对786-O细胞药物敏感性的影响。罗丹明123实验检测细胞P糖蛋白(P-gp)功能,Western blot方法检测经典多药耐药基因(mdr1)产物P-gp的表达。结果在0.00001~0.1mmol/L浓度范围内,H2O2具有明显的促细胞生长作用,且显著增加了阿霉素及长春新碱的细胞毒性,0.02mmol/LH2O2孵育786-O细胞72h后其对阿霉素及长春新碱的药物敏感性分别增加至对照组的5.43及4.47倍,荧光染料罗丹明123的蓄积量显著增加,Western blot检测结果显示0.02mmol/LH2O2可抑制肾癌786-O细胞P-gp的表达。结论低浓度活性氧H2O2可部分逆转人肾癌786-O细胞先天性多药耐药对阿霉素的耐药性,其逆转机制与增加细胞内化疗药物浓度、抑制P-gp的表达有关。 相似文献
17.
Megumi Ueno Minako Nyui Ikuo Nakanishi Kazunori Anzai Toshihiko Ozawa Ken-ichiro Matsumoto Yoshihiro Uto 《Journal of Clinical Biochemistry and Nutrition》2014,54(2):75-80
The scavenging activity of rat plasma against hyperthermia-induced reactive oxygen species was tested. The glutathione-dependent reduction of a nitroxyl radical, 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl, which was restricted by adding superoxide dismutase or by deoxygenating the reaction mixture, was applied to an index of superoxide (O2•−) generation. A reaction mixture containing 0.1 mM 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl and 1 mM glutathione was prepared using 100 mM phosphate buffer containing 0.05 mM diethylenetriaminepentaacetic acid. The reaction mixture was kept in a screw-top vial and incubated in a water bath at 37 or 44°C. The time course of the electron paramagnetic resonance signal of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl in the reaction mixture was measured by an X-band EPR spectrometer (JEOL, Tokyo, Japan). When the same experiment was performed using rat plasma instead of 100 mM PB, the glutathione-dependent reduction of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl, i.e., generation of O2•−, was not obtained. Only the first-order decay reduction of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl, which indicates direct reduction of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl, was obtained in rat plasma. Adding 0.5% albumin to the phosphate buffer reaction mixture could almost completely inhibit O2•− generation at 37°C. However, addition of 0.5% albumin could not inhibit O2•− generation at 44°C, i.e., hyperthermic temperature. Ascorbic acid also showed inhibition of O2•− generation by 0.01 mM at 37°C, but 0.02 mM or more could inhibit O2•− generation at 44°C. A higher concentration of ascorbic acid showed first-order reduction, i.e., direct one-electron reduction, of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl. Hyperthermia-induced O2•− generation in rat plasma can be mostly inhibited by albumin and ascorbic acid in the plasma. 相似文献
18.
目的:探讨白细胞精子症不育患者精液中白细胞(WBC)密度与活性氧(ROS)和尿酸(UA)之间的关系。方法:依据WHO诊断标准,选择白细胞精子症不育者45例,非白细胞精子症不育者35例,生育者30例,采用过氧化物酶染色法进行精液WBC密度计数;用硫代巴比妥酸(TBA)显色法测定丙二醛(MDA)值;采用尿酸酶-过氧化物酶偶联法测定UA含量。结果:白细胞精子症组精液MDA值[(20.795±8.132)μmol/L]、WBC计数[(1.785±0.686)×109/L]显著高于生育组MDA值[(8.415±3.673)μmol/L]、WBC计数[(0.038±0.024)×109/L],而UA含量[(146.9±67.1)μmol/L]低于生育组UA含量[(398.6±52.3)μmol/L](P<0.01)。结论:白细胞精子症不育患者精液ROS产生增多,致抗氧化物UA含量下降,使精子中毒受损。提示临床在治疗时应加用抗氧化药物,可提高疗效。 相似文献