尿酸对血管内皮细胞氧化应激反应的影响及其对细胞的损伤作用

发布时间：2017-12-31 03:48

本文关键词：尿酸对血管内皮细胞氧化应激反应的影响及其对细胞的损伤作用　出处：《中南大学》2012年博士论文　论文类型：学位论文

【摘要】：目的观察尿酸(UA)对人血管内皮细胞氧化应激反应、以及细胞凋亡与坏死的影响。方法体外培养人脐静脉血管内皮细胞(HUVEC),以0、0.05、0.10、0.20、0.30g/L浓度UA分别作用6、12、24及48h,利用流式细胞仪检测HUVEC细胞凋亡率、细胞坏死率、以及活性氧(ROS)含量；采用分光光度计检测HUVEC的超氧阴离子(02-)含量；应用ELISA法检测HUVEC培养上清液中还原型酰胺腺嘌呤二核苷酸磷酸(NADPH)、黄嘌呤氧化酶(XO)、内皮型一氧化氮合酶(eNOS)、环加氧酶-2(COX-2)、脂加氧酶(LOX)水平；同时检测分别使用NADPH抑制剂DPI、XO抑制剂Allo、eNOS抑制剂eNOSR后上述指标的变化。结果 1.HUVEC与不同浓度UA(0、0.05、0.10、0.20、0.30g/L)共培养不同时间(6、12、24、48h)后,细胞凋亡率显著性增加,且细胞凋亡率随着UA浓度增加和作用时间延长而显著上升；当UA浓度为0.30g/L、以及作用时间为48h时,细胞坏死率显著性增加。 2.HUVEC与不同浓度UA(0、0.05、0.10、0.20、0.30g／L)共培’养不同时间(6、12、24、48h)后,细胞内ROS及02-表达显著性增加,且随着刺激浓度增加和作用时间延长而显著上升；当UA浓度为0.30g/L、以及作用时间为48h时,细胞内ROS及02-表达明显下降；ROS及02-表达量与HUVEC细胞凋亡率呈正相关关系。 3.HUVEC与不同浓度UA(0、0.05、0.10、0.20、0.30g/L)共培养24h后,NADPH、XO及eNOS表达量随着UA浓度增加而显著增加,其表达量与HUVEC细胞凋亡率高度正相关；而COX-2及LOX表达量无明显变化,其表达量与HUVEC细胞凋亡率不相关。 4.UA与不同氧化酶抑制剂共刺激HUVEC24h后,UA可激活NADPH、XO、eNOS表达,DPI抑制NADPH及eNOS表达,Allo抑制XO及eNOS表达,eNOSR抑制eNOS表达,且DPI、Allo及eNOSR均可降低ROS及02-表达；UA+DPI+Allo组ROS及02-表达较UA+Allo组显著性降低,但较UA+DPI组无显著性差异；UA+DPI+eNOSR组ROS及02-表达较UA+DPI组及UA+eNOS组均显著性降低。结论 1.UA呈一定浓度依赖性和时间依赖性促进HUVEC损伤。 2.UA主要通过活化氧化酶NADPH及eNOS从而激活氧化应激反应途径导致HUVEC损伤。
[Abstract]:objective
To observe the effect of uric acid (UA) on oxidative stress in human vascular endothelial cells, and the effect of apoptosis and necrosis.
Human umbilical vein endothelial cells (HUVEC) cultured in vitro, with the concentration of 0,0.05,0.10,0.20,0.30g/L UA and 48h 6,12,24 respectively, using HUVEC flow cytometry to detect apoptosis rate, cell necrosis rate and reactive oxygen species (ROS) with superoxide anion content; Spectrophotometric Determination of HUVEC (02-) was detected by HUVEC ELISA; method in the supernatant reduced nicotinamide adenine dinucleotide phosphate (NADPH), xanthine oxidase (XO), endothelial nitric oxide synthase (eNOS), cyclooxygenase -2 (COX-2), Lipoxygenase (LOX) level; at the same time were detected by NADPH inhibitor DPI, XO inhibitor Allo, the changes of the above indicators the eNOS inhibitor eNOSR.
Result
1.HUVEC and UA of different concentrations (0,0.05,0.10,0.20,0.30g/L) were cultured for different time (6,12,24,48h), the apoptosis rate was significantly increased, and the apoptosis rate with the increase of UA concentration and the prolongation of time increased significantly; when the concentration of UA was 0.30g/L, and the reaction time was 48h, cell necrosis rate was significantly increased.
2.HUVEC with different concentrations of UA (0,0.05,0.10,0.20,0.30g / L) co culture "raised in different time (6,12,24,48h), intracellular expression of ROS and 02- were significantly increased, and with the stimulation of the concentration and time increased significantly; when the concentration of UA was 0.30g/L, and the reaction time was 48h, the expression of ROS and 02- cells decreased significantly ROS; and 02- expression and apoptosis rate of HUVEC cells was positively correlated.
3.HUVEC with different concentrations of UA (0,0.05,0.10,0.20,0.30g/L) co culture 24h, NADPH, XO and eNOS expression with UA concentration increased, and the expression of the apoptosis rate of HUVEC cells is highly correlated; but no significant change of COX-2 and LOX expression, and the expression of HUVEC cell apoptosis rate.
With different 4.UA oxidase inhibitors were stimulated HUVEC24h, UA can activate NADPH and XO, the expression of eNOS, DPI and eNOS Allo inhibited the expression of NADPH, inhibit the expression of XO and eNOS, eNOSR inhibits the expression of eNOS, and DPI, Allo and eNOSR can reduce the expression of 02- and ROS; ROS and 02- expression in UA+DPI+Allo group was significantly lower than that in group UA+Allo however, compared with the UA+DPI group had no significant difference between the expression of UA+DPI+eNOSR and 02-; ROS group compared with UA+DPI group and UA+eNOS group were significantly lower.
conclusion
1.UA has a certain concentration dependence and time dependence to promote HUVEC damage.
2.UA is mainly activated by activation oxidase NADPH and eNOS to activate the oxidative stress reaction pathway to cause HUVEC damage.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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