【摘要】：背景與目的缺血性心臟病是我國目前致死率和致殘率最高的疾病之一。雖然及時恢復缺血心肌血流灌注是治療的重要手段,但心肌缺血后最終心肌梗死面積大約一半以上是由缺血再灌注損傷(ischemia reperfusion injury,IRI)所致。心肌缺血再灌注過程中局部微環(huán)境產(chǎn)生的緩激肽、腺苷、氫離子等可興奮心肌缺血敏感性傳入神經(jīng)元,并將傷害感受信號經(jīng)背根神經(jīng)節(jié)(dorsal root ganglia,DRG)和相應節(jié)段脊髓上傳至高級中樞。脊髓背角淺層尤其是脊髓背角II層又稱膠狀質(zhì)SG(substantia gelatinosa,SG),是傷害性信息向中樞傳遞的初級門戶,主要由興奮性中間神經(jīng)元組成,對傷害性刺激信息的傳入與整合具有重要的調(diào)控作用。心肌缺血引起相應節(jié)段脊髓神經(jīng)元興奮,進而通過交感反射釋放神經(jīng)遞質(zhì)加重心肌細胞損傷。因此,調(diào)控脊髓背角神經(jīng)元興奮性,抑制神經(jīng)遞質(zhì)的釋放,可能會減輕缺血性心肌損傷。本課題組前期的研究發(fā)現(xiàn),鞘內(nèi)嗎啡預處理(intrathecal morphine preconditioning,ITMP)可明顯減輕心肌缺血后損傷。然而,ITMP對遠端心臟發(fā)揮保護作用的神經(jīng)機制尚未闡明,本研究擬探討ITMP對抗心肌IRI的神經(jīng)信號調(diào)控機制。方法SD雄性大鼠,體重180~280 g,成功建立鞘內(nèi)置管模型。本實驗分兩個系列進行研究。第一部分:探討ITMP對大鼠心肌缺血后脊髓SG神經(jīng)元興奮性的神經(jīng)調(diào)控機制。實驗隨機分為6組(n=6),假手術(shù)組(SHAM組)、缺血再灌注組(IR組)、鞘內(nèi)嗎啡預處理組(ITMP組)、δ受體阻斷劑NTD+ITMP組(NTD+ITMP組)、κ受體阻斷劑nor-BNI+ITMP組(nor-BNI+ITMP組)和μ受體阻斷劑CTOP+ITMP組(CTOP+ITMP組)。所有組除SHAM組外均以結(jié)扎冠狀動脈左前降支缺血30 min、再灌注120 min的方法建立心肌缺血再灌注損傷模型。ITMP組在心肌缺血前30 min經(jīng)鞘內(nèi)泵注嗎啡(3μg·kg-1,10μl)5 min,停止5 min,共3個循環(huán);I/R組給予等容量生理鹽水。NTD+ITMP組、nor-BNI+ITMP組以及CTOP+ITMP組分別于嗎啡預處理前10 min鞘內(nèi)分別注射NTD(1 ug·ul-1,10 ul)、nor-BNI(1 ug·ul-1,10 ul)、CTOP(1 ug·ul-1,10 ul)。于再灌注10 min結(jié)束時,急性分離大鼠T2~T6脊髓,制備脊髓組織切片,采用全細胞膜片鉗技術(shù)對大鼠SG神經(jīng)元進行動作電位(action potential,AP)記錄,包括SG神經(jīng)元靜息電位(resting potential,RP)、動作電位閾值(threshold of action potential,APT)、動作電位峰值(peak of action potential,APP)、動作電位時程[用半峰時間(APD50)表示],記錄步階電流40、60、80、100 p A誘發(fā)的動作電位個數(shù)。第二部分:研究ITMP對心肌缺血后損傷及相關(guān)神經(jīng)遞質(zhì)釋放的影響。實驗分組以及動物模型建立同第一部分研究。實驗過程中記錄心律失常的發(fā)生率。于再灌注120 min時處死大鼠,取心肌組織,測定梗死區(qū)(IS)體積與缺血危險區(qū)(ARR)面積,計算IS/ARR比值;Western blot法檢測T2~T6脊髓和DRG組織中c-fos蛋白表達;免疫熒光觀察T2~T6脊髓背角P物質(zhì)(substance P,SP)、降鈣素基因相關(guān)肽(calcitonin gene related peptide,CGRP)和內(nèi)嗎啡肽2(endomorphin 2,EM-2)的釋放水平以及DRG中的SP和CGRP的表達情況。結(jié)果第一部分:探討ITMP對大鼠心肌缺血后脊髓背角SG神經(jīng)元興奮性的神經(jīng)調(diào)控機制結(jié)果發(fā)現(xiàn),心肌缺血后大鼠脊髓背角SG神經(jīng)元興奮性增強,表現(xiàn)為APT降低,APP增加,相同步階電流刺激電流下誘發(fā)的AP個數(shù)增多;而ITMP顯著逆轉(zhuǎn)IR組大鼠SG神經(jīng)元的興奮性升高,表現(xiàn)為APT升高,APP降低,相同步階電流刺激下誘發(fā)的AP個數(shù)減少。各組大鼠脊髓背角SG神經(jīng)元RP和APD50差異無統(tǒng)計學意義。與ITMP組相比,通過預先分別給予δ、κ、μ三種阿片受體阻斷劑可以取消SG神經(jīng)元興奮性的降低,表現(xiàn)為APT降低,APP增高,相同步階刺激下誘發(fā)的AP個數(shù)增加。第二部分:研究ITMP對心肌缺血后損傷及相關(guān)神經(jīng)遞質(zhì)釋放的影響1.ITMP介導的大鼠心肌保護效應:ITMP可明顯降低心肌梗死體積和心律失常發(fā)生率,且NTD、nor-BNI和CTOP完全阻斷ITMP的心肌保護作用。2.ITMP對大鼠心肌缺血后脊髓SP、CGRP和EM-2表達水平的影響:免疫熒光結(jié)果顯示,SP、CGRP和EM-2免疫陽性產(chǎn)物在脊髓背角淺層存在共表達。心肌缺血后脊髓背角SP和CGRP大量表達,ITMP可以顯著抑制心肌缺血后脊髓背角SP和CGRP的表達上調(diào);EM-2在心肌缺血后脊髓背角的表達無明顯變化,而ITMP可以顯著上調(diào)心肌缺血后EM-2的表達。3.ITMP對大鼠心肌缺血后DRG內(nèi)SP和CGRP表達水平的影響:免疫熒光結(jié)果顯示心肌缺血后,DRG內(nèi)SP和CGRP表達均顯著增加,ITMP可顯著減少心肌缺血再灌注損傷誘導的DRG內(nèi)SP和CGRP的表達增加。4.ITMP對大鼠心肌缺血后脊髓背角和DRG內(nèi)c-fos表達的影響:Western blot結(jié)果證明,心肌缺血后大鼠脊髓背角和DRG內(nèi)c-fos明顯增多,ITMP可顯著抑制心肌缺血后大鼠脊髓背角和DRG內(nèi)c-fos表達的增多;三種中樞阿片受體阻斷劑能取消ITMP的抑制效應。結(jié)論ITMP對在體大鼠心肌缺血后損傷具有保護作用。其可能的神經(jīng)調(diào)控機制:1.ITMP心肌保護作用與降低心肌缺血后大鼠脊髓SG神經(jīng)元的興奮性,減輕心臟傷害感受反應,調(diào)控脊髓水平傷害感受信號有關(guān),δ、κ、μ三種中樞阿片受體均參與了這種保護作用。2.ITMP心肌保護作用與抑制心肌缺血后脊髓SP和CGRP等神經(jīng)遞質(zhì)的過度釋放,促進EM-2在脊髓水平的釋放,降低神經(jīng)元興奮性有關(guān)。
[Abstract]:BACKGROUND & OBJECTIVE Ischemic heart disease is one of the most lethal and disabled diseases in China at present. Although timely recovery of ischemic myocardial perfusion is an important means of treatment, more than half of the final myocardial infarction area after myocardial ischemia is caused by ischemia reperfusion injury (IRI). Bradykinin, adenosine and hydrogen ions produced in the local microenvironment during reperfusion excite myocardial ischemia-sensitive afferent neurons and upload nociceptive signals via dorsal root ganglia (DRG) and corresponding segments of the spinal cord to the higher central nervous system. Gelatinosa, SG, is the primary portal of nociceptive information to the central nervous system. It is mainly composed of excitatory intermediate neurons. It plays an important role in regulating the input and integration of nociceptive stimulus information. Myocardial ischemia induces excitation of spinal neurons in corresponding segments, and then releases neurotransmitters through sympathetic reflex to aggravate myocardial injury. Previous studies have shown that intrathecal morphine preconditioning (ITMP) can significantly reduce myocardial ischemic injury. However, ITMP plays a protective role in the distal heart. Methods SD male rats, weighing 180-280 g, were successfully established intrathecal catheterization model. This study was divided into two series. Part I: To investigate the neural regulation mechanism of ITMP on the excitability of spinal SG neurons after myocardial ischemia in rats. Six groups (n=6), sham operation group (SHAM group), ischemia-reperfusion group (IR group), intrathecal morphine preconditioning group (ITMP group), delta receptor blocker NTD+ITMP group (NTD+ITMP group), kappa receptor blocker nor-BNI+ITMP group (nor-BNI+ITMP group) and mu receptor blocker CTOP+ITMP group (CTOP+ITMP group). All groups except SHAM group were ligated with 30 mi left anterior descending coronary artery ischemia. The model of myocardial ischemia-reperfusion injury was established by reperfusion for 120 minutes.Morphine was injected intrathecally 30 minutes before myocardial ischemia in ITMP group for 5 minutes and stopped for 5 minutes.The same volume of saline was given to I/R group.NTD+ITMP group,nor-BNI+ITMP group and CTOP+ITMP group were injected intrathecally 10 minutes before morphine preconditioning respectively. NTD (1 ug.ul-1,10 ul), nor-BNI (1 ug.ul-1,10 ul), CTOP (1 ug.ul-1,10 ul). At the end of 10 minutes of reperfusion, the T2-T6 spinal cord of rats was separated and the spinal cord tissue sections were prepared. The action potential (AP) of SG neurons, including resting potential (R-P) of SG neurons, were recorded by whole-cell patch clamp technique. P, threshold of action potential (APT), peak of action potential (APP), action potential duration (expressed as APD50), and the number of action potentials evoked by step current 40, 60, 80, 100 P A were recorded. Part II: To study the effects of ITMP on myocardial ischemia injury and related neurotransmitter release. The incidence of arrhythmia was recorded during the experiment. Rats were sacrificed at 120 minutes after reperfusion. The infarct area (IS) and the area of ischemic risk area (ARR) were measured and the ratio of IS to ARR was calculated. The expression of c-fos protein in spinal cord and DRG tissues of T2-T6 was detected by Western blot. The release levels of substance P (SP), calcitonin gene related peptide (CGRP) and endomorphin 2 (EM-2) and the expression of SP and CGRP in DRG were observed by immunofluorescence. Results Part I: To investigate the excitability of ITMP on SG neurons in spinal dorsal horn after myocardial ischemia in rats. The results showed that the excitability of SG neurons in spinal dorsal horn was enhanced after myocardial ischemia, which was manifested by the decrease of APT, the increase of APP and the increase of the number of AP induced by synchronous current stimulation, while ITMP significantly reversed the increase of excitability of SG neurons in IR group, which was manifested by the increase of APT, the decrease of APP and the induction of synchronous current stimulation. There was no significant difference in RP and APD50 of SG neurons in the spinal dorsal horn of each group. Compared with ITMP group, the decreased excitability of SG neurons could be cancelled by pretreatment with delta, kappa, and mu opioid receptor blockers respectively. The decrease of APT, the increase of APP and the increase of AP induced by synchronous stimuli were observed. Effects of ITMP on myocardial ischemia injury and related neurotransmitter release 1. ITMP-mediated myocardial protective effect: ITMP can significantly reduce myocardial infarction volume and arrhythmia incidence, and NTD, nor-BNI and CTOP completely block the myocardial protective effect of ITMP. 2. ITMP on the expression of SP, CGRP and EM-2 in spinal cord after myocardial ischemia in rats Immunofluorescence showed that SP, CGRP and EM-2 immunoreactive products co-expressed in the superficial layer of the spinal dorsal horn. After myocardial ischemia, SP and CGRP were overexpressed in the spinal dorsal horn. ITMP could significantly inhibit the up-regulation of SP and CGRP expression in the spinal dorsal horn after myocardial ischemia. EM-2 expression in the spinal dorsal horn after myocardial ischemia did not change significantly, but ITMP could significantly up-regulate the Regulation of EM-2 expression after myocardial ischemia. 3. Effect of ITMP on SP and CGRP expression in DRG after myocardial ischemia in rats: Immunofluorescence results showed that SP and CGRP expression in DRG increased significantly after myocardial ischemia. ITMP could significantly reduce the expression of SP and CGRP in DRG induced by myocardial ischemia-reperfusion injury. Effects of three central opioid receptor blockers on the expression of c-fos in dorsal horn and DRG: Western blot showed that the expression of c-fos in spinal dorsal horn and DRG increased significantly after myocardial ischemia in rats. ITMP significantly inhibited the expression of c-fos in spinal dorsal horn and DRG after myocardial ischemia in rats. Postoperative neuroprotective effects of ITMP on myocardial ischemia are related to decreasing excitability of spinal SG neurons, reducing cardiac nociceptive response, and regulating nociceptive signals at spinal cord level. Three central opioid receptors, delta, kappa and mu, are involved in this protective effect. 2. The protective effect is related to inhibiting the excessive release of neurotransmitters such as SP and CGRP in the spinal cord after myocardial ischemia, promoting the release of EM-2 at the spinal cord level and reducing the excitability of neurons.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R614

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