【作者】 贾强用；

【导师】 石刚刚；

【作者基本信息】 汕头大学 ， 药理学， 2007， 硕士

【摘要】 Egr-1是即早基因家族的一员，能在众多刺激因素的作用下快速表达，介导细胞对外界环境改变的反应。Egr-1在心脏排斥反应，肺缺血再灌注（I／R）损伤中起到了中心性的作用。但Egr-1在心肌缺血再灌注中的作用尚未明确，故而课题组应用Egr-1反义寡核苷酸在整体和离体两方面观察了Egr-1在心肌缺血再灌注及心肌细胞缺氧复氧（H／R）中的作用。发现Egr-1在心脏I／R及心肌细胞H／R中起到了非常重要的作用。课题组合成的化合物碘化N-正丁基氟哌啶醇（F₂）能够抑制缺血再灌注心肌Egr-1mRNA及蛋白的表达，同时观察到了心脏功能的改善。可见F₂可通过抑制Egr-1mRNA及蛋白的表达起到拮抗心肌I／R损伤的作用。那么F₂通过抑制Egr-1表达，减轻I／R损伤的作用是否是F₂所用作用机制呢?先前的研究已经表明Egr-1在心肌I／R中起到了非常重要的作用，有研究发现作为细胞内重要的信使Ca²⁺、PKC参与了对Egr-1的调控。然而，有关Egr-1在心肌I／R下高表达的分子机制尚未明确。因此本研究主要围绕二个方面进行探讨：①在应用Egr-1反义寡核苷酸的基础上加用F₂，通过观察Egr-1蛋白表达及心脏功能的变化来探讨这一问题。②采用原代培养的心肌细胞H／R模型，运用离体心肌细胞H／R损伤模拟在体心肌I／R损伤，应用维拉帕米，PKC抑制剂H7、BIM探讨Ca²⁺／PKC信号通路在心肌细胞Egr-1表达中的作用。方法1．心肌缺血再灌注模型的建立及实验分组：手术结扎大鼠冠状动脉左前降支造成心肌缺血60min后解除结扎再灌注180min。大鼠随机分成五组：对照（Control）组、缺血再灌注（I／R）组、溶剂（PEG）组、反义寡核苷酸（AS）组、反义寡核苷酸+F₂（AS+F₂）组。实验结束后，取结扎线下缺血心肌组织，用Western blot法测定心肌组织中Egr-1蛋白表达情况；比色法测定大鼠血清肌酸激酶（CK）、乳酸脱氢酶（LDH）的活性和大鼠心肌组织髓过氧化物酶（MPO）的活性；硫代巴比妥酸法测定心肌组织丙二醛（MDA）的含量；黄嘌呤氧化酶法测定心肌组织超氧化物歧化酶（SOD）的活性。实验过程中监测血流动力学，全程记录心率（HR）、收缩压（SP）、舒张压（DP）、左心室收缩压（LVSP）、左心室压变化速率最大值（±dp／dtmax）。2．心肌细胞缺氧复氧模型的建立及实验分组：进行原代心肌细胞培养，细胞生长至5-7d后，对其进行随机分组：对照（Control）组、缺氧复氧（H／R）组、溶剂（DMSO）组、EGTA组、维拉帕米（VER）组、H7组和BIM组。H／R组细胞先换用被高纯度氮气饱和的缺氧液，置于缺氧箱中37℃密闭培养3h，后又继续在正常培养基中按常规培养条件培养1h，造成H／R损伤。RT-PCR法检测培养心肌细胞中Egr-1mRNA的表达水平。Western-blot法检测培养心肌细胞中Egr-1蛋白的表达水平。结果1．AS、AS+F₂对Egr-1蛋白水平的影响与Control组比，I／R组Egr-1蛋白表达明显增强（P＜0.05）；与I／R组比，AS组及AS+F₂组Egr-1蛋白表达明显减弱（P＜0.05）。AS+F₂组与AS组比无统计学意义（P＞0.05）。2．AS、AS+F₂对心肌组织中MPO、SOD活性和MDA含量的影响与I／R组比，AS、AS+F₂能有效降低缺血再灌注心肌组织中MPO的活性（P＜0.05），MDA的含量（P＜0.05），保护SOD的活性（P＜0.05）。AS+F₂组与AS组比有统计学意义（P＜0.05）。3．AS、AS+F₂对血清中LDH、CK活性的影响与Control组比，I／R组血清中LDH、CK的活性明显提高（P＜0.05）。与I／R组比，AS组、AS+F₂组心肌酶的释放明显降低（P＜0.05）。与AS组比，AS+F₂组心肌酶的释放明显降低。4．AS、AS+F₂对血流动力学的影响缺血期，各组HR、SP、DP、LVSP、±dp／dtmax均降低，与I／R组比，AS组及AS+F₂组有统计学意义（P＜0.05），AS+F₂组与AS组比无统计学意义（P＞0.05）：再灌注时，与I／R组比，AS组和AS+F₂组有统计学意义（P＜0.05），AS+F₂组与AS组比，有统计学意义（P＜0.05）。5．VER、EGTA、H7、BIM对缺氧复氧心肌细胞Egr-1表达的影响5．1．VER、EGTA、H7、BIM对培养心肌细胞Egr-1 mRNA表达水平的影响：RT-PCR结果显示：与Control组相比，H／R组及DMSO组心肌细胞Egr-1mRNA的表达水平明显增高；与H／R组比，VER组、EGTA组、H7组、BIM组Egr-1 mRNA的表达明显下调，差异具有统计学意义（P＜0.05）。5．2．VER、EGTA、H7、BIM对培养心肌细胞Egr-1蛋白表达水平的影响：Western blot结果显示：与Control组相比，H／R组及DMSO组心肌细胞Egr-1mRNA的表达水平明显增高；与H／R组比，VER组、EGTA组、H7组、BIM组Egr-1蛋白的表达明显下调，差异具有统计学意义（P＜0.05）。结论1)F₂能通过抑制Egr-1的过量表达起到抗心肌缺血再灌注的作用：F₂除通过抑制Egr-1起到抗心肌缺血再灌注损伤外还有其它机制参与。2)Ca²⁺／PKC信号通路参与了心肌细胞缺氧复氧中Egr-1的高表达。更多还原

【Abstract】 Early growth response-1 （Egr-1）, a transcription factor and an immediate early gene, can beinduced in a transient manner by a variety of extracellular stimuli. The over expression of Egr-1was the crucial mechanism in heart graft rejective reaction and lung ischemia-reperfusion injury.But the mechanism of Egr-1 in I/R remains unclear. Use antisense oligonucleotides（AS） in vivoand in vitro, found that Egr-1 was the main mechanism in heart I/R injury. N-n-butylHaloperidol Iodide （F₂） could supress the Egr-1 mRNA and protein expression. Does all theprotection of F₂ on myocardial I/R injury through suppressing Egr-1 expression. Previous studyhas found that Ca²⁺、PKC participate in the regulation of Egr-1.But the mechanism of Egr-1expression in I/R remains unclear. So this research focuses on two problems.①Use AS, AS+ F₂in cultured cardiomyocytes, view the Egr-1 protein expression and the heart function.②Useverapamil, PKC specific inhibitor H7, BIM in cultured cardiomyocytes after H/R to investigateCa²⁺/PKC Pathway.Methods1. The rat experimental models in vivo （I/R） were established by 60 min left anteriordescending coronary artery （LAD） occlusion followed by 180 min of reperfusion. All ratswere randomly assigned into one of five groups: Control, I/R, PEG, AS and AS+ F₂.Hemodynamics was monitored with BL420 system. The expression levels of Egr-1 proteinin myocardium were examined by Western-blot. Levels of CK,LDH in the serum and MPOin the myocardium were mesured by ehromatometry. Levels of MDA in the myocardium were mesured by thiobarbituric acid method. Levels of SOD in the myocardium weremesured by xanthine oxidase method.2. Primary culture of neonatal rat cardiomyocytes and preparation of H/R model: After beingreplaced the initial culture medium with hypoxic buffer, the cardiomyocytes were incubatedin an air-tight chamber gassed with pure N₂ for 3 h of hypoxia. The buffer was then replacedwith fresh oxygenated culture medium and the dishes were transferred into a normoxicincubator for 1 h of reoxygenation. The cardiomyocytes were randomly divided into one offollowing groups: Control, H/R, DMSO, EGTA, VER, H7 or BIM. The expression levels ofEgr-1 mRNA in cultured cardiomyocytes were examined by RT-PCR. The expression levelsof Egr-1 protein in cultured cardiomyocytes were examined by Western-blot.Results1. The effect of AS, AS+ F₂ on Egr-1 protein expression in myocardiumRelative to the Control group, Ischemia-reperfusion induced a significantly increase inEgr-1 protein expression in myocardium（P＜0.05）. Compared with the I/R group, levels ofEgr-1 protein measured by Western blot analysis in myocardium in the group AS and AS+F₂ were significantly decreased at the end of experiment（P＜0.05）. Compare to the AS group,These changes in levels of Egr-1 protein were not altered by F2 （P＞0.05）.2. The effect of AS, AS+ F₂ on MPO, SOD and MDA in myocardiumRelative to the I/R group, levels of MPO, MDA in myocardium in the group AS and AS+F2were significantly decreased（P＜0.05 ）. levels of SOD were increased（P＜0.05 ）. Compare tothe AS group, these changes in levels of levels of MPO, MDA, SOD were strengthened.3. The effect of AS, AS+F₂ on CK, LDH activities in serumIschemia-reperfusion induced a significantly increase in CK, LDH activities in serum（P＜0.05）. Compared with I/R group, AS group and AS+F₂ group reduced CK and LDHactivity（P＜0.05）. Compared with AS group, AS+F₂ group reduced CK and LDH activity（P＜0.05）.4. The effect of AS, AS+F₂ on hemodynamicsValues of HR, SP, DP, LVSP,±dp/dtmax were significantly lower in I/R hearts during ischemia period. Compared with I/R group, values of HR, SP, DE LVSP,±dp/dtmax werehigher in AS and AS+F₂ groups, But there was no significant difference between the twogroups. Compared with AS group,Values of SP, DP, LVSP, +dp/dtmax during reperfusionwere higher in AS+F₂ groups.5. The effect of VER, EGTA, H7 or BIM on Egr-1 expression in cultured cardiomyocytes5.1 Relative to the Control group, levels of Egr-1 rnRNA measured by RT-PCR analysisin cultured cardiomyocytes in the H/R group were significantly increased at the end ofexperiment （P＜0.05）. These changes in levels of Egr-1 mRNA were not altered by DMSO,but significantly reduced when VER, EGTA, H7 or BIM was added into cultured cells （P＜0.05）.5.2 Consistent with changes in levels of Egr-1 mRNA in the H/R group, Egr-1 proteinmeasured by Western blot analysis in cultured cardiomyocytes were significantly increasedrelative to the Control group （P＜0.05）. These changes in levels of Egr-1 protein were notaltered by DMSO, but significantly reduced when treated with VER, EGTA, H7 or BIM （P＜0.05）.Conclusions（1） F₂ can protect myocardium from ischemia-reperfusion by inhibiting Egr-1 overexpression,but that is not the whole mechanisms of F₂.（2） Ca²⁺/PKC sigal pathway involved in hypoxia-reoxygen induced Egr-1 gene expression incultured cardiomyocytes.更多还原