长期压力超负荷心力衰竭大鼠心肌间质、交感神经重塑及PKC激活的意义

发布时间：2018-01-30 06:03

  本文关键词： 心脏压力超负荷 心力衰竭 交感神经密度 去甲肾上腺素转运蛋白 心肌间质 蛋白激酶C epsilon　出处：《河北医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：多种心血管病，如高血压、心脏瓣膜病、心肌梗死和心肌病等均导致心力衰竭（heart failure，HF），HF导致心血管病患者病残、死亡的重要原因，HF发生、进展机制一直是研究的重点。众所周知，在HF发生、进展过程中，心脏交感神经功能（cardiac sympathetic nerve function,CSNF）异常发挥重要作用。研究证据表明，心血管病中的CSNF异常，包括交感神经活性（sympathetic nerve activity，SNA）和/或再摄取功能（reuptake）变化与心肌间质重塑有密切关系，但确切机制尚不完全清楚。逐渐积累的研究证据还显示，发挥多种细胞信号调控作用重要的信号分子蛋白激酶C epsilon（protein kinase C，PKCε）激活，不但可介导神经生长、调控神经突触功能，还参与诱发心肌间质纤维化。由此，我们推测，PKCε激活可能同时对HF的心肌间质重塑和CSNF，特别是再摄取功能异常发挥重要影响。本实验通过建立以心肌纤维化为特征，同时伴有交感神经再摄取功能下降的心脏压力超负荷（pressure overload，POL）HF动物模型，观察动物模型HF时心肌PKCε亚型激活与心脏交感神经再摄取功能蛋白膜表达与心肌间质重塑的关系，并讨论PKCε激活可能对此发挥的作用及意义。 方法：30只7周雄性Wistar大鼠，按随机计数表法分为2组：假手术组（SHAM）（n=15）和POL组（n=15）。适应性饲养一周后，以苯巴比妥%腹腔麻醉，用腹主动脉缩窄方法建立HF动物模型。模型建立8周后，检测心脏血流动力学变化，大鼠固定后，麻醉，，行颈动脉插管，将导管置于左心室，待状态稳定后，记录有创血流动力学的各项指标：左心室收缩压（left ventricular systolic pressure、LVSP）、左心室舒张末压（leftventricular end-diastolic pressure、LVEDP）、左心室压力变化最大速率（maxmaximal rate of rise of left ventricular pressure and max maximal rate ofdecline of left ventricular pressure、±dp/dtmax），检测完毕，生理盐水灌流，摘取心脏及双侧颈交感神经节，分装入冻存管，于液氮罐内保存。 各组动物心肌组织HE染色后，用显微镜检测左心室心肌细胞形态。用分子生物学方法检测去甲肾上腺素转运蛋白及mRNA表达：RT-PCR法检测颈交感神经节中NET的mRNA表达；免疫组织化学方法检测交感神经标志物--酪氨酸羟化酶（tyrosine hydroxylase，TH）及新生神经标记物--生长相关蛋白（Growth Associated Protein43，GAP43）表达；WesternBlot法检测心脏组织中NET和PKCε细胞膜及胞浆中蛋白表达；由图像分析系统（Image Pro Plus6.0）处理各组大鼠心肌组织图像，用SPSS13.0软件进行各组数据的统计学分析，P＜0.05为差异有统计学意义。 结果： 1心脏血流动力学检测：POL组大鼠左室收缩压（LVSP）降低（P＜0.05）,左室舒张末压（LVDEP）升高（P＜0.05），左心室压力变化最大速率（±dp/dtmax）降低（P＜0.05），表明POL组大鼠发生心力衰竭，处于失代偿期。 2心脏和体重重量比值（Heart Weight/Body Weight，HW/BW）：POL组是SHAM组的1.34倍（P＜0.05），表明长期心脏POL诱导心脏肥大，并观察到POL组的左室腔明显缩小，室壁增厚。 3放免法测定血浆去甲肾上腺素浓度：POL组较SHAM组显著增加（P＜0.05）。 4高倍显微镜观测心肌细胞形态：与SHAM组比较，POL组心肌细胞长度和直径增加（P＜0.05）。 5心肌间质胶原变化：Sirius Red染色及偏光镜观察到，心肌间质及血管周围胶原I及III型均增加，且以I型胶原增加明显，图像分析显示POL组间质胶原容积分数（ICVF）为SHAM组1.69倍（P＜0.05）。 6心脏交感神经密度及神经新生：与SHAM组比较，POL组心肌TH蛋白及GAP43蛋白，表达增加（P＜0.05）。 7PKC激活与NET膜表达：Western Blot结果显示：与SHAM组比较，POL组心肌细胞膜与胞浆中NET蛋白量比值降低（P＜0.05），说明其NET内化明显；PKCε蛋白的细胞膜与胞浆比值增高（P＜0.05），则说明POL组的PKCε活化增多。 8心脏神经节NET mRNA表达： RT-PCR结果显示，对照组与POL组大鼠神经节中NET mRNA表达无统计学差异。 结论： 长期心脏POL可导致： 1向心性心肌细胞肥厚，心脏重量增加，心肌间质纤维化，心功能下降； 2PKCε蛋白激活，，蛋白膜表达增加，同时（1）心脏交感神经密度，新生神经增加；（2）交感神经NET蛋白膜表达降低，但对心脏神经节NET mRNA表达水平无影响； 实验结果说明：长期心脏POL可诱导PKCε蛋白激活，同时伴心肌纤维化、交感神经重塑，交感神经NET膜表达降低，提示PKCε信号途径激活，可能在这些变化中发挥重要的中介作用。
[Abstract]:Objective: many cardiovascular diseases, such as hypertension, valvular heart disease, myocardial infarction and cardiomyopathy have led to heart failure (heart failure, HF, HF) lead to disability in patients with cardiovascular disease, HF an important reason, death, progress mechanism has been the focus of the study. It is well known that in HF, the progress of the process, the function of cardiac sympathetic nerve (cardiac sympathetic nerve function, CSNF) abnormalities play an important role. Research evidence that cardiovascular disease CSNF abnormalities, including sympathetic nerve activity (sympathetic nerve, activity, SNA) and / or reuptake function (reuptake) and the change of myocardial interstitial remodeling is closely related, but the exact mechanism is not entirely clear. Research evidence accumulation also showed that play a variety of cell signaling regulation of important signaling molecules of protein kinase C (epsilon protein kinase C, PKC E) activation, not only mediates nerve growth regulation. Synaptic function also plays an important role in inducing myocardial interstitial fibrosis. Thus, we speculate that PKC may also activate epsilon HF on myocardial interstitial remodeling and CSNF, especially the reuptake dysfunction plays an important role. Through this experiment to establish myocardial fibrosis is characterized with sympathetic reuptake function decreased heart pressure load (pressure overload, POL HF) animal model, observe the animal model of myocardial HF PKC epsilon isoform of cardiac sympathetic nerve activation and reuptake function protein expression and membrane remodeling the relationship between myocardium, and discuss the role and significance of PKC epsilon activation might play.
Methods: 30 7 week old male Wistar rats, randomly count were divided into 2 groups: sham operation group (SHAM) (n=15) and POL group (n=15). After one week of adaptive feeding, with phenobarbital% intraperitoneal anesthesia, narrow method to establish HF animal model with abdominal aorta model for 8 weeks. After the detection of cardiac hemodynamic changes in rats after fixation, anesthesia, carotid artery intubation, left ventricular catheter placed in the stable state, to record the indicators, hemodynamics, left ventricular systolic pressure (left ventricular systolic pressure, LVSP), left ventricular end diastolic pressure (LEFTVENTRICULAR end-diastolic Shi Shuzhang pressure. LVEDP), maximum rate of left ventricular pressure change (maxmaximal rate of rise of left ventricular pressure and Max maximal rate ofdecline of left ventricular pressure + dp/dtmax), detection after saline perfusion, the removal of the heart and cervical sympathetic The ganglion is divided into the cryopreservation tube and stored in the liquid nitrogen tank.
Each animal myocardial HE staining, microscope detection of left ventricular myocardial cell morphology. Using molecular biology methods for detecting the norepinephrine transporter and the expression of mRNA: to detect the expression of RT-PCR in cervical sympathetic ganglion NET mRNA; immunohistochemistry of sympathetic marker tyrosine hydroxylase (tyrosine hydroxylase, TH) and the new nerve markers of growth associated protein (Growth, Associated, Protein43, GAP43) expression; protein expression of NET and PKC - cell membrane and heart tissue were detected by WesternBlot cell cytoplasm; analysis system consists of image processing (Image Pro Plus6.0) in myocardial tissue of rats in each group were analyzed by the statistical data of each image, using SPSS13.0 software, P < 0.05 the difference was statistically significant.
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