巯基受损在内皮细胞NO系统功能障碍中的作用及其机制初探

发布时间：2018-06-14 16:59

本文选题：谷胱甘肽(GSH) + 马来酸二乙酯(DEM)　；参考：《四川大学》2006年硕士论文

【摘要】：目的：通过巯基螯合剂DEM对内皮细胞二甲基精氨酸二甲胺水解酶(DDAH)活性的影响，探讨氧化应激导致NO通路功能障碍的可能机制及其在动脉粥样硬化发病中的作用。方法：在培养的内皮细胞中加入不同剂量DEM、作用不同时间，以确定能明显降低细胞内GSH含量但却不明显影响细胞存活率的最适DEM作用浓度和作用时间。以该剂量DEM预处理内皮细胞30分钟后去除DEM，换成无血清培养基继续培养0、0.5、2、6、12、24小时后，检测各时间段的细胞内GSH含量、DDAH活性及上清液中NO含量。实验按照6个不同的检测点分成6大组，分别为0、0.5、2、6、12、24小时组。分别设对照组与加药组。细胞存活率检测用MTT法；细胞内GSH含量用荧光测定法；细胞内DDAH活性用分光光度法检测与ADMA反应后生成的L-瓜氨酸的生成量来表示；上清液中NO含量用NO试剂盒检测。结果：MTT结果显示DEM最佳的作用浓度为0.6μmol／L，作用时间为30分钟，该浓度DEM处理内皮细胞30分钟后，GSH含量已明显降低，但内皮细胞生存率仅比对照组轻微下降，未明显影响细胞存活率；GSH浓度的检测显示，经DEM预处理后半小时细胞内GSH含量已较对照组明显降低(降低28.7％，p＜0.05)，随后逐步回升，12小时反超正常水平，随后回落，至24小时时恢复至正常水平；DDAH活性的检测显示，经DEM预处理后DDAH活性随之降低，至2小时时达最低值(降低80.07％，，p＜0.01)，以后DDAH活性逐渐恢复，24小时后亦出现反超，呈类似于GSH
[Abstract]:Aim: to investigate the possible mechanism of oxidative stress induced no pathway dysfunction and its role in the pathogenesis of atherosclerosis through the effect of mercapto chelating agent Dem on the activity of dimethyl arginine dimethylamine hydrolase (DDAH) in endothelial cells. Methods: different doses of DEM were added to cultured endothelial cells for different time to determine the optimal concentration and duration of Dem which could significantly reduce the content of GSH in the cells but did not affect the survival rate of the cells. After the endothelial cells were pretreated with Dem for 30 minutes, DEM was replaced by serum-free culture medium for 24 hours. After 24 hours, GSH content and DDAH activity and no content in supernatant were measured. According to six different detection points, the experiment was divided into 6 groups. They were divided into two groups: control group and drug addition group. The cell viability was detected by MTT assay, the intracellular GSH content was measured by fluorescence assay, the activity of DDAH was measured by spectrophotometry, and the production of L-citrulline was measured by spectrophotometry, and the no content in supernatant was detected by no kit. Results the optimal concentration of Dem was 0.6 渭 mol / L and the time of exposure was 30 minutes. The GSH content of endothelial cells was significantly decreased after 30 minutes of Dem treatment, but the survival rate of endothelial cells was only slightly decreased compared with the control group. The concentration of GSH did not affect the survival rate of the cells. The results showed that the GSH content in the cells treated with Dem was significantly lower than that in the control group at half hour after Dem preconditioning (lower than 28.7p < 0.05), then gradually increased to the normal level for 12 hours, then fell back to normal level. The detection of DDAH activity returned to normal level at 24 hours showed that after Dem pretreatment, the activity of DDAH decreased and reached the lowest value at 2 hours (80.07% lower than 0. 01%). After 24 hours, the activity of DDAH also appeared reverse ultrasound, which was similar to that of GSH.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363

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