本文選題：動(dòng)脈壓力反射　切入點(diǎn)：ENaC　出處：《鄭州大學(xué)》2017年碩士論文

【摘要】：背景介紹心力衰竭(Heart failure,HF)簡(jiǎn)稱心衰,是各種心臟疾病,比如冠心病、高血壓以及慢性腎臟病等各種心血管疾病發(fā)展的終末階段。壓力反射(baroreflex)又稱減壓反射,是心血管活動(dòng)調(diào)節(jié)的重要機(jī)制之一。位于主動(dòng)脈弓和頸動(dòng)脈竇的感覺(jué)神經(jīng)末梢感受動(dòng)脈血壓的變化,經(jīng)減壓神經(jīng)和竇神經(jīng)傳入,到達(dá)心血管中樞,通過(guò)調(diào)節(jié)交感神經(jīng)和迷走神經(jīng)的功能,平衡心血管活動(dòng)。1969年Smyth等人首次以Smyth法測(cè)定BRS(baaroreflex sensitivity)來(lái)表示壓力反射的敏感性。心衰動(dòng)物的壓力反射敏感性明顯降低,擾亂自主神經(jīng)平衡,導(dǎo)致交感神經(jīng)興奮性增強(qiáng),副交感神經(jīng)活動(dòng)降低,從而使發(fā)病率和死亡率增加。據(jù)文獻(xiàn)報(bào)道,減壓反射的傳入神經(jīng)敏感性和興奮性降會(huì)低導(dǎo)致減壓反射敏感性降低,但至今尚不清楚傳入神經(jīng)對(duì)血壓變化敏感性降低的機(jī)制。研究發(fā)現(xiàn)HF引起Na+通道功能降低抑制了減壓神經(jīng)的電活動(dòng),而在減壓神經(jīng)末梢上存在著感受血壓變化的機(jī)械敏感性離子通道,心衰可能是改變了這種機(jī)械敏感性離子通道的功能,降低減壓神經(jīng)對(duì)血壓變化的敏感性。上皮鈉通道(Epithelial Sodium Channel,ENa C)屬于ENa C/Deg家族,具有阿米洛利敏感性,是該家族中目前研究最為廣泛的分子。ENa C是由α、β、γ、δ四種亞基組成的異源多聚體蛋白,主要分布于腎臟等上皮組織中,功能是維持水,鈉平衡。通過(guò)醛固酮與細(xì)胞內(nèi)的醛固酮受體結(jié)合,增加ENa C通道在細(xì)胞膜上的表達(dá),調(diào)節(jié)ENa C介導(dǎo)的Na+細(xì)胞轉(zhuǎn)運(yùn)。在神經(jīng)節(jié)等非上皮組織中,ENa C是機(jī)械敏感性離子通道。發(fā)生心力衰竭(HF)時(shí)醛固酮顯著升高,升高正常動(dòng)物或者人血液中的醛固酮顯著降低減壓反射的敏感性。已有實(shí)驗(yàn)發(fā)現(xiàn)減壓神經(jīng)末梢和胞體中有ENa C通道蛋白的分布,而對(duì)醛固酮是否影響減壓神經(jīng)中ENa C通道及其可能機(jī)制尚未報(bào)道。目的本實(shí)驗(yàn)?zāi)M心衰引起大鼠機(jī)體醛固酮濃度升高,觀察減壓反射敏感性變化,以及結(jié)狀神經(jīng)節(jié)的減壓神經(jīng)胞體中ENa C表達(dá)變化,以確定血漿醛固酮升高是否影響減壓神經(jīng)中ENa C通道的表達(dá)。方法1、采用滲透泵持續(xù)灌輸醛固酮的方法,制作高醛固酮?jiǎng)游锬Ｐ�。體重200 g的SD雄性大鼠在10%的水合氯醛腹腔麻醉狀態(tài)下,于腹背后皮下植入Alzet2004微型滲透泵。2、30只健康雄性SD大鼠給予正常飼料,適應(yīng)喂養(yǎng)1周后,隨機(jī)分為5組,均喂養(yǎng)4周。所有的大鼠在20%的烏拉坦(0.5 ml/100 g)腹腔麻醉下,(1)正常對(duì)照組(CON,n=6):正常飲食和飲水。(2)溶劑組(VEH,n=6):正常飲食和飲水,裝有5%甲醇的滲透泵。(3)螺內(nèi)酯組(SPI,n=6):正常飲食和飲水的基礎(chǔ)上,每天以25 mg/kg灌胃。(4)醛固酮組(ALD,n=6):正常飲食和飲水,將裝有溶解在5%甲醇的醛固酮的滲透泵,埋于大鼠腹背部皮下。(5)醛固酮+螺內(nèi)酯組(ALD+SPI,n=6):正常攝食基礎(chǔ)上,皮下植入滲透泵,泵中裝有溶解在5%甲醇中的醛固酮。醛固酮(0.75μg/h)或僅5%甲醇經(jīng)微量滲透泵持續(xù)皮下恒流輸注。螺內(nèi)酯(25 mg/kg/day)經(jīng)灌胃。3、測(cè)量血漿醛固酮濃度:模型制備后,于實(shí)驗(yàn)前,取SD大鼠的動(dòng)脈血液1.5-2ml。離心機(jī)以1000 g離心15分鐘,去出上清液,放置-80℃保存。使用大鼠醛固酮試劑盒測(cè)量大鼠血漿內(nèi)醛固酮的濃度。4、血壓和心率的測(cè)量:用20%的烏拉坦5 ml/kg腹腔麻醉,使用股動(dòng)脈插管技術(shù),測(cè)量血壓和心率,每只動(dòng)物測(cè)量3次,取其收縮壓的平均值作為基礎(chǔ)值。5、動(dòng)脈壓力反射敏感性測(cè)定:各組動(dòng)物于4周后,參照文獻(xiàn)按照改良的Smyth方法進(jìn)行動(dòng)脈壓力反射敏感性檢測(cè)。股靜脈插管,通過(guò)股靜脈給藥,注射硝普鈉(SNP)和苯腎上腺素(PE)分別誘發(fā)升壓反射和降壓反射,以反射前后的收縮壓變化值與心動(dòng)周期變化值之比(ΔSBP/ΔCC)作為升壓反射敏感性(BRS-SNP)和降壓反射敏感性(BRS-PE)指標(biāo)。6、取五組大鼠的結(jié)狀神經(jīng)節(jié)(NG),采用免疫組織化學(xué)技術(shù)檢測(cè)ENa C-β、ENa C-γ的表達(dá),并使用ENa C-β、ENa C-γ與Neu N蛋白標(biāo)志的免疫熒光共染色,鑒別ENa C-β、ENa C-γ在結(jié)狀神經(jīng)節(jié)(NG)中的分布。7、4周后取五個(gè)組大鼠的結(jié)狀神經(jīng)節(jié)(NG)組織,采用Western blot分別檢測(cè)脊結(jié)狀神經(jīng)節(jié)(NG)中的ENa C-β和ENa C-γ表達(dá)及變化。8、統(tǒng)計(jì)學(xué)處理:實(shí)驗(yàn)數(shù)據(jù)以均值±標(biāo)準(zhǔn)差(mean±SD)表示,采用Graph Pad Prism5.0統(tǒng)計(jì)軟件,采用單因素方差分析和t檢驗(yàn),P㩳0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果1.給醛固酮4周后,大鼠體內(nèi)醛固酮550.9±7.63 pg/ml,明顯高于對(duì)照組(193.2±4.95 pg/ml,P㩳0.001)。2.升高醛固酮后,大鼠血壓(98.14±3.76 mm Hg)、心率(357.8±8.32 beat/min)、體重(412.7±7.65 g)與對(duì)照組相比(94.65±3.97 mm Hg,345.7±11.59 beta/min,416.8±10.3 g),無(wú)明顯改變(P㧐0.05)。3.醛固酮升高后,大鼠減壓反射敏感性(BRS)(0.225±0.05 ms/mm Hg)比正常組(1.113±0.04)ms/mm Hg)的明顯降低(P㩳0.001)。而醛固酮受體阻斷劑螺內(nèi)酯(0.973±0.05 ms/mm Hg)可以阻斷醛固酮對(duì)BRS的影響組,使之恢復(fù)近正常水平。4.免疫熒光雙染確定ENaC-β和ENa C-γ蛋白在NenN標(biāo)記的神經(jīng)元胞體(結(jié)狀神經(jīng)節(jié))處有表達(dá)。5.通過(guò)滲透泵維持大鼠機(jī)體高醛固酮濃度,采用western blot檢測(cè)發(fā)現(xiàn):醛固酮組的結(jié)狀神經(jīng)節(jié)中表達(dá)β/γENa C蛋白,正常組為100,ENa C-β和ENa C-γ蛋白的表達(dá)量分別為31.51±3.33和27.10±3.52(P㩳0.001)比正常組明顯減少,而醛固酮+螺內(nèi)酯合用組的β/γENa C的表達(dá)量分別為97.08±10.08和98.05±11.14(P㧐0.05)與正常組比較,基本恢復(fù),表明醛固酮受體阻斷劑螺內(nèi)酯阻斷了醛固酮對(duì)ENa C的表達(dá)。結(jié)論正常大鼠體內(nèi),ALD升高,通過(guò)與受體途徑降低減壓反射神經(jīng)元胞體中ENa C表達(dá),減弱壓力反射。
[Abstract]:Background heart failure (Heart failure HF) is referred to as heart failure, heart diseases, such as coronary artery disease, end stage chronic kidney disease and hypertension and other cardiovascular disease. Baroreflex (baroreflex) also called depressor reflex, is one of the important mechanisms regulating cardiovascular activity. Changes of sensory nerve endings in the aortic arch and carotid artery sinus feeling of arterial blood pressure, after decompression of nerve and sinus nerve afferent, reach the cardiovascular center, through the regulation of sympathetic nerve and vagus nerve function, cardiovascular activity.1969 Smyth balance for the first time in the determination of BRS by Smyth (baaroreflex sensitivity) to represent the baroreflex sensitivity. Baroreflex sensitivity was significantly reduced heart failure animal, disrupting autonomy nerve balance, leading to enhanced excitability of sympathetic nerve and parasympathetic nerve activity decreased, thereby increasing morbidity and mortality. According to the text Reported, afferent nerve sensitivity and excitability of the depressor reflex low lead depressor reflex sensitivity decreased, but it is still not clear on the mechanism of nerve afferent blood pressure reduced susceptibility. The study found that the Na+ channel function is reduced to inhibit the electrical activity of depressor nerve caused by HF, the mechanosensitive ion channels and the change of blood pressure is felt in vacuum on nerve endings, heart failure may be changed this mechanosensitive ion channel function, reduces the sensitivity to changes in blood pressure depressor nerve. The epithelial sodium channel (Epithelial Sodium Channel, ENa C ENa) belongs to the C/Deg family, with amiloride sensitive in the family, is currently the most widely studied molecular.ENa C by alpha beta, gamma, delta four heterologous subunits of multimeric protein mainly distributed in kidney epithelial tissues, function is to maintain the water and sodium balance through aldosterone and fine. Combined with the intracellular mineralocorticoid receptor, increases the expression of ENa C channel in the cell membrane, regulate ENa mediated by C Na+ cells in the ganglion. Other non epithelial tissues, ENa C is a mechanosensitive ion channel. The occurrence of heart failure (HF) when aldosterone increased significantly increased in normal animals or human blood aldosterone significantly reduce the sensitivity. The depressor reflex experiments indicated that the distribution of ENa C protein in decompression nerve terminals and cell bodies, and whether the effects of ENa on aldosterone C nerve decompression channel and its possible mechanism has not yet been reported. The purpose of this experiment simulated the body caused by heart failure aldosterone concentration in rats was observed and ENa depressor reflex sensitivity, C the nodose ganglion neurons in the expression of decompression, to determine whether elevated plasma aldosterone on expression of ENa in C channel depressor nerve. Methods 1, using osmotic pump continuous irrigation 杈撻啗鍥洪叜鐨勬柟娉，

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