本文選題：辛伐他汀　切入點(diǎn)：缺氧復(fù)氧　出處：《蘇州大學(xué)》2006年博士論文　論文類型：學(xué)位論文

【摘要】：目的探討缺氧復(fù)氧對血管內(nèi)皮細(xì)胞t-PA、PAI-1、NO和NOS表達(dá)的影響及辛伐他汀的干預(yù)作用,并探討其可能的機(jī)制。 方法體外培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞株ECV304,使用自制的缺氧小室對ECV304進(jìn)行缺氧復(fù)氧處理,將細(xì)胞培養(yǎng)于24孔培養(yǎng)板和25ml培養(yǎng)瓶。1.將ECV304細(xì)胞進(jìn)行缺氧復(fù)氧處理,分別于缺氧2小時(shí)、復(fù)氧2、4、8、16小時(shí)收集所需標(biāo)本,觀察缺氧復(fù)氧對t-PA、PAI-1、NO、NOS活力及eNOSmRNA、iNOSmRNA、eNOS蛋白、表達(dá)的影響;2.不同濃度的辛伐他汀(0.1、1.0、5.0、10.0μmol/L)以及10.0μmol/L的辛伐他汀+0.2mmol/L的甲羥戊酸處理ECV304細(xì)胞24小時(shí)后再行缺氧2小時(shí)復(fù)氧2小時(shí)處理,收集所需標(biāo)本,觀察辛伐他汀對t-PA、PAl-1、NO和NOS活力及eNOSmRNA、iNOSmRNA、eNOS蛋白表達(dá)的影響; 3.用10.0μmol/L的辛伐他汀預(yù)處理ECV304 24小時(shí)后再行缺氧2小時(shí)、復(fù)氧2、4、8、16小時(shí)處理,收集各時(shí)間段細(xì)胞培養(yǎng)液,觀察辛伐他汀預(yù)處理對ECV304分泌NO和NOS活力的影響。4.不同濃度的辛伐他汀(0.1、1.0、10.0μmol/L)以及10.0μmol/L的辛伐他汀+0.2 mmol/L的甲羥戊酸處理ECV304細(xì)胞24小時(shí),收集各組細(xì)胞標(biāo)本,提取總RNA和胞漿蛋白,觀察辛伐他汀對eNOSmRNA、iNOSmRNA、eNOS蛋白表達(dá)的影響。測定培養(yǎng)液中t-PA、PAI-1濃度用ELISA法;檢測培養(yǎng)液中NO的含量及NOS活力分別用硝酸還原酶法和化學(xué)比色法。收集25ml培養(yǎng)瓶細(xì)胞,進(jìn)行RT-PCR檢測iNOS、eNOS基因相對表達(dá)量,采用異硫青酸-苯酚-氯仿一步法提取細(xì)胞總RNA,純度檢驗(yàn)合格后,取總RNA做逆轉(zhuǎn)錄生成cDNA,進(jìn)行半定量PCR,產(chǎn)物經(jīng)圖象分析系統(tǒng)掃描評價(jià)各組iNOS和eNOS基因相對表達(dá)量;Western-blots檢測eNOS蛋白相對表達(dá)量,以上各組細(xì)胞提取總蛋白,進(jìn)行SDS-聚丙烯酰胺凝膠電泳,再進(jìn)行電轉(zhuǎn)膜,用一抗和二抗進(jìn)行免疫印跡反應(yīng),顯色,圖象掃描分析,檢測eNOS蛋白相對表達(dá)量。 結(jié)果1.復(fù)氧2小時(shí)、4小時(shí)培養(yǎng)液中tPA濃度明顯增加(P均0.01),缺氧2小時(shí)、復(fù)氧2、4小時(shí):PAI-1濃度明顯增加(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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R363;R96

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