缺氧复氧对血管内皮细胞t-PA、PAI-1、NO及NOS表达的影响及辛伐他汀的干预研究

发布时间：2018-03-03 00:02

本文选题：辛伐他汀　切入点：缺氧复氧　出处：《苏州大学》2006年博士论文　论文类型：学位论文

【摘要】：目的探讨缺氧复氧对血管内皮细胞t-PA、PAI-1、NO和NOS表达的影响及辛伐他汀的干预作用,并探讨其可能的机制。方法体外培养人脐静脉内皮细胞株ECV304,使用自制的缺氧小室对ECV304进行缺氧复氧处理,将细胞培养于24孔培养板和25ml培养瓶。1.将ECV304细胞进行缺氧复氧处理,分别于缺氧2小时、复氧2、4、8、16小时收集所需标本,观察缺氧复氧对t-PA、PAI-1、NO、NOS活力及eNOSmRNA、iNOSmRNA、eNOS蛋白、表达的影响;2.不同浓度的辛伐他汀(0.1、1.0、5.0、10.0μmol/L)以及10.0μmol/L的辛伐他汀+0.2mmol/L的甲羟戊酸处理ECV304细胞24小时后再行缺氧2小时复氧2小时处理,收集所需标本,观察辛伐他汀对t-PA、PAl-1、NO和NOS活力及eNOSmRNA、iNOSmRNA、eNOS蛋白表达的影响; 3.用10.0μmol/L的辛伐他汀预处理ECV304 24小时后再行缺氧2小时、复氧2、4、8、16小时处理,收集各时间段细胞培养液,观察辛伐他汀预处理对ECV304分泌NO和NOS活力的影响。4.不同浓度的辛伐他汀(0.1、1.0、10.0μmol/L)以及10.0μmol/L的辛伐他汀+0.2 mmol/L的甲羟戊酸处理ECV304细胞24小时,收集各组细胞标本,提取总RNA和胞浆蛋白,观察辛伐他汀对eNOSmRNA、iNOSmRNA、eNOS蛋白表达的影响。测定培养液中t-PA、PAI-1浓度用ELISA法;检测培养液中NO的含量及NOS活力分别用硝酸还原酶法和化学比色法。收集25ml培养瓶细胞,进行RT-PCR检测iNOS、eNOS基因相对表达量,采用异硫青酸-苯酚-氯仿一步法提取细胞总RNA,纯度检验合格后,取总RNA做逆转录生成cDNA,进行半定量PCR,产物经图象分析系统扫描评价各组iNOS和eNOS基因相对表达量;Western-blots检测eNOS蛋白相对表达量,以上各组细胞提取总蛋白,进行SDS-聚丙烯酰胺凝胶电泳,再进行电转膜,用一抗和二抗进行免疫印迹反应,显色,图象扫描分析,检测eNOS蛋白相对表达量。结果1.复氧2小时、4小时培养液中tPA浓度明显增加(P均0.01),缺氧2小时、复氧2、4小时:PAI-1浓度明显增加(P均0.05);5.0、10.0μmol/L辛
[Abstract]:Objective to investigate the effects of hypoxia and reoxygenation on the expression of no and NOS in vascular endothelial cells (VEC) and the intervention of simvastatin and its possible mechanism. Methods Human umbilical vein endothelial cell line ECV304 was cultured in vitro. ECV304 cells were treated with anoxic reoxygenation chamber. The cells were cultured in 24-well culture plate and 25ml culture flask. ECV304 cells were treated with anoxia reoxygenation for 2 hours, respectively. Samples were collected for 16 hours after reoxygenation. The NOS activity of t-PAI-1 and eNOSmRNA-iNOSmRNA-Enos protein were observed, and the effects of anoxia and reoxygenation on the activity of nitric oxide synthase (NOS) and the expression of eNOSmRNA-iNOSmRNA-Enos protein were observed. Different concentrations of simvastatin (0.1 渭 mol / L) and simvastatin 0.2 mmol / L (10.0 渭 mol / L) and simvastatin 0.2 mmol / L methylvalerate (10.0 渭 mol / L) were treated with anoxia for 2 hours and reoxygenated for 2 hours. To observe the effect of simvastatin on the activity of no and NOS and the expression of eNOSmRNA-iNOSmRNA-Enos protein in t-PA-PAl-1nmRNA.3.After pretreatment with simvastatin of 10.0 渭 mol/L for 24 hours, ECV304 was treated with anoxia for 2 hours and reoxygenation for 816 hours, and the cell culture fluid was collected. To observe the effect of simvastatin pretreatment on the activity of no and NOS in ECV304. The effects of different concentrations of simvastatin 0.1 渭 mol / L and 10.0 渭 mol/L simvastatin 0.2 mmol/L mevalic acid on the activity of no and NOS were observed in ECV304 cells for 24 hours. The total RNA and cytosolic protein were extracted from the samples of each group. To observe the effect of simvastatin on the expression of eNOSmRNA-iNOSmRNA-Enos protein, to determine the concentration of t-PAPPAI-1 in culture medium by ELISA method, to detect the content of no and the activity of NOS by nitrate reductase method and chemical colorimetry, respectively. The relative expression of iNOS gene was detected by RT-PCR, and the total RNAs were extracted by isothiocyanate-phenol-chloroform one-step method. The relative expression of iNOS and eNOS genes in each group was evaluated by image analysis system. The relative expression of eNOS protein was detected by Western-blots. SDS-polyacrylamide gel electrophoresis (SDS-polyacrylamide gel electrophoresis), electroporation membrane, immunoblotting reaction with first antibody and second antibody, color development, image scanning analysis, and detection of relative expression of eNOS protein were carried out. Results: 1. The concentration of tPA increased significantly in the culture medium for 2 hours and 4 hours after reoxygenation. The concentration of tPA in the medium increased significantly (P 0.01), hypoxia for 2 hours, and reoxygenation for 2 hours for 4 hours. The concentration of tPA increased significantly at 0.05 渭 mol/L (5.0 渭 mol/L). 2 hours after reoxygenation, the concentration of PAI-1 increased significantly. 2.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R363;R96

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