本文選題：強(qiáng)啡肽　切入點(diǎn)：к阿片受體　出處：《第四軍醫(yī)大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 阿片肽和阿片受體在體內(nèi)廣泛分布,長(zhǎng)期以來(lái)人們一直認(rèn)為阿片肽是一種中樞神經(jīng)肽或神經(jīng)遞質(zhì),僅在中樞神經(jīng)系統(tǒng)發(fā)揮作用。近20年來(lái)研究發(fā)現(xiàn)心臟也可合成內(nèi)源性阿片肽,通過(guò)自分泌或旁分泌的方式對(duì)自身功能進(jìn)行調(diào)節(jié),其作用是通過(guò)各自相應(yīng)的阿片受體所介導(dǎo)的。與心臟功能密切相關(guān)的阿片受體主要是μ、δ和к三種亞型,其中在心血管系統(tǒng)占主導(dǎo)地位的阿片受體亞型是к阿片受體,強(qiáng)啡肽(Dyn)是к阿片受體的內(nèi)源性配體。 研究證實(shí),心肌缺血預(yù)處理(IPC)能夠使心臟對(duì)隨后的持續(xù)的心肌缺血再灌注(I/R)損傷的耐受性提高,IPC對(duì)缺血心肌的這種保護(hù)作用主要是由于缺血觸發(fā)釋放內(nèi)源性物質(zhì)實(shí)現(xiàn)的。已知在正常條件下,心臟可合成Dyn并能激活κ阿片受體產(chǎn)生對(duì)心血管系統(tǒng)的調(diào)節(jié)作用;在心肌缺血時(shí),內(nèi)源性Dyn有可能釋放增多并通過(guò)激動(dòng)κ阿片受體參與心肌缺血的病理過(guò)程。因此本試驗(yàn)旨在研究?jī)?nèi)源性Dyn是否在IPC心肌保護(hù)作用中扮演重要的角色以及激活κ阿片受體是否對(duì)缺血再灌注心肌具有直接的保護(hù)作用。 1、目的: (1)觀察在IPC過(guò)程中,Dyn在血漿水平的濃度變化以及Dyn在心肌組織mRNA表達(dá)的變化。 (2)給以κ阿片受體的選擇性激動(dòng)劑,觀察其對(duì)大鼠心肌I/R損傷的影響。 (3)給以κ阿片受體的選擇性阻斷劑,觀察其對(duì)IPC的心肌保護(hù)作用的影響。 2、研究方法: (1) IPC模型制備方法:大鼠冠狀動(dòng)脈左前降支(LAD)距左心耳下緣約2 mm處穿線,套扎LAD,心肌缺血5 min,松開(kāi),再灌注5 min,3個(gè)循環(huán)。 (2) I/R模型制備方法:套扎LAD,心肌缺血45 min,松開(kāi),再灌注120 min。 (3)應(yīng)用生理學(xué)整體實(shí)驗(yàn)技術(shù)進(jìn)行大鼠血液的采集和心臟功能的測(cè)定。 (4)采用RT-PCR方法觀察大鼠心肌組織Dyn mRNA表達(dá)的變化。 (5)采用放射免疫分析方法測(cè)定血漿中Dyn,磷酸肌酸激酶(CK)和乳酸脫氫酶(LDH)的水平。 (6)應(yīng)用雙重染色法檢測(cè)大鼠心肌梗死面積。 3、主要結(jié)果: (1)采集各組大鼠血液,離心取上層血漿,測(cè)定各組大鼠血漿中Dyn的濃度,IPC組(缺血5 min,再灌注5 min,3個(gè)循環(huán)后采樣)的濃度顯著高于同時(shí)間sham組(不給予缺血和再灌注,其余處理同IPC組)(P0.05);IPC組的濃度顯著高于con組(未給予任何處理)(P0.05)。 (2)取大鼠心肌組織,觀察各組心肌組織Dyn mRNA表達(dá)的變化,IPC組(缺血5 min,再灌注5 min,3個(gè)循環(huán)后采樣)強(qiáng)啡肽基因表達(dá)水平與同時(shí)間sham組(不給予缺血和再灌注,其余處理同IPC組)和con組(未給予任何處理)相比,無(wú)顯著差別(P0.05)。 (3)靜脈給予選擇性κ阿片受體激動(dòng)劑U50488H(1.5 mg/kg)發(fā)現(xiàn),U50488H+I/R組大鼠心肌梗死面積明顯低于I/R組的大鼠心肌梗死面積(P0.01);血漿中CK和LDH的含量也明顯降低(P0.01)。靜脈給予選擇性κ阿片受體拮抗劑Nor-BNI(1.5 mg/kg),Nor-BNI+U50488H+I/R組大鼠與I/R組大鼠心肌梗死范圍無(wú)顯著性差異(P0.05),血漿中CK和LDH的含量也無(wú)顯著性差異(P0.05),但與U50488H+I/R組大鼠心肌梗死范圍及血漿中CK和LDH相比,明顯升高(P0.01)。 (4)各組以缺血30 min開(kāi)始時(shí)為記錄起始點(diǎn),分別記錄缺血0 min,缺血10 min,缺血30 min,再灌注30 min時(shí)間點(diǎn)的左心室收縮壓(LVSP)及收縮功能(dp/dt_(max))和舒張功能(-dp/dt_(max)),發(fā)現(xiàn)IPC+I/R組在缺血10 min,缺血30 min,再灌注30 min時(shí)間點(diǎn)的LVSP和±dp/dt_(max)明顯高于同時(shí)間點(diǎn)的I/R組(P0.05)。靜脈給予κ阿片受體的特異性阻斷劑Nor-BNI(2 mg/kg),Nor-BNI+IPC+I/R組在缺血10 min,缺血30 min,再灌注30 min時(shí)間點(diǎn)的LVSP和±dp/dt_(max)明顯低于IPC+I/R組(P0.05)。 4、結(jié)論: (1)本研究首次表明,IPC可能促進(jìn)機(jī)體內(nèi)源性阿片肽Dyn的釋放而增加了血漿Dyn水平,后者可通過(guò)激活κ阿片受體產(chǎn)生對(duì)心臟的保護(hù)作用。 (2)外源性阿片類物質(zhì)U50488H激活κ阿片受體,能夠顯著降低I/R的心肌梗死面積,減少心肌細(xì)胞內(nèi)蛋白酶的漏出,對(duì)I/R心肌具有直接保護(hù)作用。 (3)給予κ阿片受體的特異性阻斷劑Nor-BNI,能夠阻斷IPC對(duì)于I/R心肌在收縮功能方面的改善,進(jìn)一步證明κ阿片受體介導(dǎo)了IPC對(duì)I/R心肌的保護(hù)作用。
[Abstract]:Opioid and opioid receptors are widely distributed in the body, it has long been recognized that opioid peptide is a kind of central neuropeptides or neurotransmitters, only play a role in the central nervous system. In the past 20 years, the study found that the heart can also synthesize endogenous opioid peptides by autocrine or paracrine regulation of their function, its role is through the respective opioid receptor mediated by opioid receptors. Closely related with the heart function is mainly u, Delta and kappa three subtypes, which dominates in the cardiovascular system of opioid receptor subtype is kappa opioid receptor, dynorphin (Dyn) is an endogenous ligand of kappa opioid receptors.
The research confirmed that myocardial ischemic preconditioning (IPC) protects the heart against subsequent sustained myocardial ischemia reperfusion (I/R) injury tolerance improved, the protective effect of IPC on myocardial ischemia is mainly due to trigger the release of endogenous substances to achieve. Known in normal conditions, the heart can be synthesized and can activate Dyn kappa opioid receptor on the cardiovascular system regulation; in myocardial ischemia, endogenous Dyn may release increased and pathological process by activating kappa opioid receptors involved in myocardial ischemia. Therefore, this experiment was conducted to study whether the protective effect of endogenous Dyn in myocardial IPC plays an important role and whether the activation of kappa opioid receptor myocardial ischemia / reperfusion injury has a protective effect directly.
1, to:
(1) observed in the IPC process, the change of Dyn concentration in the plasma level of Dyn and the changes in the expression of mRNA in myocardium.
(2) with selective kappa opioid receptor agonist, to observe its effect on myocardial injury in I/R rats.
(3) with selective kappa opioid receptor antagonist, observed the effects of IPC on myocardial protection.
2, research methods:
(1) IPC model preparation methods: rat left anterior descending coronary artery (LAD) from the lower edge of the left atrial appendage about 2 mm thread ligation, LAD, myocardial ischemia 5 min, reperfusion 5 min, release the 3 cycle.
(2) I/R model preparation methods: ligation of LAD, myocardial ischemia 45 min, reperfusion 120 min. release
(3) determination of the physiological experimental technique was used to collect the blood of rats and cardiac function.
(4) to observe the changes of expression of Dyn in myocardium of mRNA rats by RT-PCR method.
(5) the plasma level of Dyn was measured by radioimmunoassay, creatine kinase (CK) and lactate dehydrogenase (LDH) level.
(6) the area of myocardial infarction was detected by double staining method.
3, the main results:
(1) blood collection rats, centrifuged supernatant plasma, plasma concentrations were measured in each group of rats Dyn, IPC group (ischemia 5 min, reperfusion 5 min, after 3 cycles of sampling) were significantly higher than sham group (not given the same time of ischemia and reperfusion, and other treatments were the same as group IPC) (P0.05); the concentration of IPC group was significantly higher than that of con group (without any treatment) (P0.05).
(2) the myocardium of rats, and observe the expression of Dyn mRNA in myocardial tissue of each group, IPC group (ischemia 5 min, reperfusion 5 min, after 3 cycles of sampling) dynorphin gene expression level and the same time sham group (not given ischemia and reperfusion, and other treatments were the same as group and con group (IPC) without any treatment) compared with no significant difference (P0.05).
(3) intravenous selective kappa opioid receptor agonist U50488H (1.5 mg/kg), the area of myocardial infarction in the rats of U50488H+I/R group was significantly lower than that in the area of myocardial infarction in I/R group (P0.01); the content of CK and LDH in plasma were significantly decreased (P0.01). Intravenous administration of a selective kappa opioid receptor antagonist Nor-BNI (1.5 mg/kg), there was no significant difference between the group Nor-BNI+U50488H+I/R rats and I/R rats myocardial infarction (P0.05), and there was no significant difference in the contents of CK and LDH in plasma (P0.05), but compared with the LDH and CK of myocardial infarct and plasma of rats in the U50488H+I/R group, was significantly increased (P0.01).
(4) each to 30 min ischemia at the start of recording starting point, were recorded 0 min ischemia, ischemia 10 min, 30 min ischemia, left ventricular systolic 30 min reperfusion time point pressure (LVSP) and systolic function (dp/dt_ (max)) and diastolic function (-dp/dt_ (max)), IPC+ in group I/R, 10 min ischemia, ischemia 30 min, reperfusion 30 min time point and LVSP + dp/dt_ (max) was significantly higher than that in group I/R at the same time point (P0.05). Intravenous opioid receptor specific antagonist Nor-BNI (2 mg/kg), group Nor-BNI+IPC+I/R in 10 min ischemia, ischemia 30 min, 30 min reperfusion time point and LVSP + dp/dt_ (max) was significantly lower than that of IPC+I/R group (P0.05).
4, conclusion:
(1) this is the first study to show that IPC may promote the endogenous opioid peptide Dyn release and increased plasma Dyn levels, which may be through the activation of kappa opioid receptor had a protective effect on the heart.
(2)澶栨簮鎬ч樋鐗囩被鐗╄川U50488H嬋，

本文編號(hào)：1638025