本文關(guān)鍵詞： 心臟壓力超負荷 心力衰竭 交感神經(jīng)密度 去甲腎上腺素轉(zhuǎn)運蛋白 心肌間質(zhì) 蛋白激酶C epsilon　出處：《河北醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：多種心血管病，如高血壓、心臟瓣膜病、心肌梗死和心肌病等均導(dǎo)致心力衰竭（heart failure，HF），HF導(dǎo)致心血管病患者病殘、死亡的重要原因，HF發(fā)生、進展機制一直是研究的重點。眾所周知，在HF發(fā)生、進展過程中，心臟交感神經(jīng)功能（cardiac sympathetic nerve function,CSNF）異常發(fā)揮重要作用。研究證據(jù)表明，心血管病中的CSNF異常，包括交感神經(jīng)活性（sympathetic nerve activity，SNA）和/或再攝取功能（reuptake）變化與心肌間質(zhì)重塑有密切關(guān)系，但確切機制尚不完全清楚。逐漸積累的研究證據(jù)還顯示，發(fā)揮多種細胞信號調(diào)控作用重要的信號分子蛋白激酶C epsilon（protein kinase C，PKCε）激活，不但可介導(dǎo)神經(jīng)生長、調(diào)控神經(jīng)突觸功能，還參與誘發(fā)心肌間質(zhì)纖維化。由此，我們推測，PKCε激活可能同時對HF的心肌間質(zhì)重塑和CSNF，特別是再攝取功能異常發(fā)揮重要影響。本實驗通過建立以心肌纖維化為特征，同時伴有交感神經(jīng)再攝取功能下降的心臟壓力超負荷（pressure overload，POL）HF動物模型，觀察動物模型HF時心肌PKCε亞型激活與心臟交感神經(jīng)再攝取功能蛋白膜表達與心肌間質(zhì)重塑的關(guān)系，并討論PKCε激活可能對此發(fā)揮的作用及意義。 方法：30只7周雄性Wistar大鼠，按隨機計數(shù)表法分為2組：假手術(shù)組（SHAM）（n=15）和POL組（n=15）。適應(yīng)性飼養(yǎng)一周后，以苯巴比妥%腹腔麻醉，用腹主動脈縮窄方法建立HF動物模型。模型建立8周后，檢測心臟血流動力學(xué)變化，大鼠固定后，麻醉，，行頸動脈插管，將導(dǎo)管置于左心室，待狀態(tài)穩(wěn)定后，記錄有創(chuàng)血流動力學(xué)的各項指標：左心室收縮壓（left ventricular systolic pressure、LVSP）、左心室舒張末壓（leftventricular end-diastolic pressure、LVEDP）、左心室壓力變化最大速率（maxmaximal rate of rise of left ventricular pressure and max maximal rate ofdecline of left ventricular pressure、±dp/dtmax），檢測完畢，生理鹽水灌流，摘取心臟及雙側(cè)頸交感神經(jīng)節(jié)，分裝入凍存管，于液氮罐內(nèi)保存。 各組動物心肌組織HE染色后，用顯微鏡檢測左心室心肌細胞形態(tài)。用分子生物學(xué)方法檢測去甲腎上腺素轉(zhuǎn)運蛋白及mRNA表達：RT-PCR法檢測頸交感神經(jīng)節(jié)中NET的mRNA表達；免疫組織化學(xué)方法檢測交感神經(jīng)標志物--酪氨酸羥化酶（tyrosine hydroxylase，TH）及新生神經(jīng)標記物--生長相關(guān)蛋白（Growth Associated Protein43，GAP43）表達；WesternBlot法檢測心臟組織中NET和PKCε細胞膜及胞漿中蛋白表達；由圖像分析系統(tǒng)（Image Pro Plus6.0）處理各組大鼠心肌組織圖像，用SPSS13.0軟件進行各組數(shù)據(jù)的統(tǒng)計學(xué)分析，P＜0.05為差異有統(tǒng)計學(xué)意義。 結(jié)果： 1心臟血流動力學(xué)檢測：POL組大鼠左室收縮壓（LVSP）降低（P＜0.05）,左室舒張末壓（LVDEP）升高（P＜0.05），左心室壓力變化最大速率（±dp/dtmax）降低（P＜0.05），表明POL組大鼠發(fā)生心力衰竭，處于失代償期。 2心臟和體重重量比值（Heart Weight/Body Weight，HW/BW）：POL組是SHAM組的1.34倍（P＜0.05），表明長期心臟POL誘導(dǎo)心臟肥大，并觀察到POL組的左室腔明顯縮小，室壁增厚。 3放免法測定血漿去甲腎上腺素濃度：POL組較SHAM組顯著增加（P＜0.05）。 4高倍顯微鏡觀測心肌細胞形態(tài)：與SHAM組比較，POL組心肌細胞長度和直徑增加（P＜0.05）。 5心肌間質(zhì)膠原變化：Sirius Red染色及偏光鏡觀察到，心肌間質(zhì)及血管周圍膠原I及III型均增加，且以I型膠原增加明顯，圖像分析顯示POL組間質(zhì)膠原容積分數(shù)（ICVF）為SHAM組1.69倍（P＜0.05）。 6心臟交感神經(jīng)密度及神經(jīng)新生：與SHAM組比較，POL組心肌TH蛋白及GAP43蛋白，表達增加（P＜0.05）。 7PKC激活與NET膜表達：Western Blot結(jié)果顯示：與SHAM組比較，POL組心肌細胞膜與胞漿中NET蛋白量比值降低（P＜0.05），說明其NET內(nèi)化明顯；PKCε蛋白的細胞膜與胞漿比值增高（P＜0.05），則說明POL組的PKCε活化增多。 8心臟神經(jīng)節(jié)NET mRNA表達： RT-PCR結(jié)果顯示，對照組與POL組大鼠神經(jīng)節(jié)中NET mRNA表達無統(tǒng)計學(xué)差異。 結(jié)論： 長期心臟POL可導(dǎo)致： 1向心性心肌細胞肥厚，心臟重量增加，心肌間質(zhì)纖維化，心功能下降； 2PKCε蛋白激活，，蛋白膜表達增加，同時（1）心臟交感神經(jīng)密度，新生神經(jīng)增加；（2）交感神經(jīng)NET蛋白膜表達降低，但對心臟神經(jīng)節(jié)NET mRNA表達水平無影響； 實驗結(jié)果說明：長期心臟POL可誘導(dǎo)PKCε蛋白激活，同時伴心肌纖維化、交感神經(jīng)重塑，交感神經(jīng)NET膜表達降低，提示PKCε信號途徑激活，可能在這些變化中發(fā)揮重要的中介作用。
[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.
Result錛

本文編號：1475492