發(fā)布時(shí)間：2019-03-27 21:14

【摘要】： 神經(jīng)肽是一類廣泛存在于機(jī)體組織且參與多種器官生理功能調(diào)節(jié)的肽類物質(zhì),其對(duì)心血管功能的影響一直是研究的熱點(diǎn),孤啡肽(nociceptin, Orphanin FQ,OFQ)即是其中之一。孤啡肽是一種結(jié)構(gòu)類似于κ阿片受體的內(nèi)源性配體-強(qiáng)啡肽A的十七肽,但它與阿片類受體的親和力很低,其特異性受體為ORL1受體,與阿片受體有很高的同源性,同屬于G蛋白耦聯(lián)受體超家族。在中樞及外周應(yīng)用孤啡肽均可引起一定程度的血壓下降和心率減慢,研究認(rèn)為除與孤啡肽參與中樞和外周神經(jīng)對(duì)心血管的調(diào)節(jié)有關(guān)外,還可能與其直接作用于心血管有關(guān)。目前在急性心肌缺血及心衰的研究中發(fā)現(xiàn)心肌與血漿中孤啡肽含量增高,提示孤啡肽可能參與上述病理過程,但是目前對(duì)其作用機(jī)制尚不清楚。孤啡肽是否對(duì)心功能有直接的調(diào)節(jié)作用?以及作用的機(jī)制如何?我們對(duì)此做了進(jìn)一步的研究。 目的:研究孤啡肽對(duì)大鼠心臟功能的影響及其可能機(jī)制,以期進(jìn)一步闡述對(duì)孤啡肽參與調(diào)節(jié)心臟生理功能的認(rèn)識(shí)。 方法:本實(shí)驗(yàn)從四個(gè)方面進(jìn)行研究 1.大鼠應(yīng)用孤啡肽(靜脈注射)的在體研究:健康大鼠分為對(duì)照組(靜脈給予等體積生理鹽水)、不同濃度孤啡肽1nM組、10nM組和50nM組,采用頸動(dòng)脈插管逆行置入左心室,觀察給藥前后LVSP、LVDP、+dP/dtmax、-dP/dtmax、HR的變化。進(jìn)一步選用孤啡肽拮抗劑UFP-101與孤啡肽配伍給藥,觀察上述指標(biāo)的變化。 2.孤啡肽及其受體的免疫熒光化學(xué)標(biāo)定實(shí)驗(yàn):采用組織免疫熒光觀察觀察孤啡肽及其受體在大鼠心肌中的分布;并且采用急性分離大鼠心肌細(xì)胞使用Confocal方法觀察孤啡肽受體的分布。 3.孤啡肽對(duì)心肌細(xì)胞的L型鈣通道、內(nèi)向整流鉀通道及瞬時(shí)外向鉀通道研究:采用膜片鉗技術(shù),觀察不同濃度的孤啡肽對(duì)上述離子通道功能的影響,以探討孤啡肽對(duì)心臟作用可能的機(jī)制。 4.孤啡肽對(duì)心肌細(xì)胞PKA活性的影響:提取分別經(jīng)不同濃度OFQ處理的心肌細(xì)胞蛋白,采用PKA活性分析試劑盒測(cè)定其活化程度,以探討孤啡肽作用可能的分子機(jī)制。 結(jié)果: 1.靜脈給予孤啡肽引起大鼠LVSP、LVDP、+dP/dtmax、-dP/dtmax、HR有明顯的劑量依賴性抑制(P0.05),預(yù)先應(yīng)用孤啡肽的拮抗劑UFP-101可以阻斷此作用。 2.免疫熒光組織化學(xué)顯示在大鼠心肌組織中有孤啡肽及其受體存在而confocal結(jié)果進(jìn)一步證實(shí)心肌細(xì)胞分布有孤啡肽受體。 3.膜片鉗實(shí)驗(yàn)結(jié)果顯示,孤啡肽對(duì)心肌細(xì)胞L型鈣電流有明顯的抑制作用,但沒有明顯的劑量依賴性,以1nM孤啡肽的作用最為明顯(抑制率為22.7%,P0.05),UFP-101可以阻斷此抑制作用。而孤啡肽對(duì)內(nèi)向整流鉀電流和瞬時(shí)外向鉀電流均沒有明顯影響。 4.心肌細(xì)胞PKA活性分析顯示,孤啡肽對(duì)心肌細(xì)胞的PKA沒有明顯影響。給予Forskolin處理心肌細(xì)胞可以明顯增高PKA活性,而孤啡肽并不能抑制這種PKA活性的增高,提示可能孤啡肽對(duì)心肌細(xì)胞的影響并不是通過PKA機(jī)制。 結(jié)論:孤啡肽對(duì)大鼠心臟左心室收縮與舒張功能有明顯的抑制作用,此作用可能是通過孤啡肽-孤啡肽受體作用,引起心肌細(xì)胞L型鈣電流的抑制所致。進(jìn)一步的研究顯示孤啡肽對(duì)于心肌細(xì)胞的PKA活性沒有影響,提示可能孤啡肽的這種作用不是通過影響cAMP-PKA通路介導(dǎo)的。對(duì)于心肌細(xì)胞的內(nèi)向整流鉀電流和瞬時(shí)外向鉀電流并未見孤啡肽對(duì)其有明顯影響。
[Abstract]:Neuropeptide is a kind of peptide substance which is widely present in the body tissue and is involved in the physiological function regulation of various organs. Its effect on the cardiovascular function has been the hot spot of the study, and the enkephalin (nociceptin, Orphanoin FQ, OFQ) is one of them. The endorphin is a 17-peptide structure similar to the endogenous ligand-dynorphin A of the opioid receptor, but its affinity to the opioid receptor is very low, and its specific receptor is the ORL1 receptor and has a high homology to the opioid receptor and is the superfamily of the G-protein-coupled receptor. In both the central and peripheral applications, the endorphins can cause a certain degree of blood pressure drop and heart rate to slow down, and the study is that, in addition to the regulation of the central and peripheral nerves involved in the central and peripheral nerves of the enkephalin, it is also possible to directly act on the cardiovascular system. At present, in the study of acute myocardial ischemia and heart failure, the content of enkephalin in the myocardium and plasma is increased, and the enkephalin may be involved in the above-mentioned pathological process, but the mechanism of its action is not clear at present. Does the enkephalin have a direct effect on the heart function? What is the mechanism of action? We made a further study of this. Objective: To study the effect of enkephalin on the heart function of rats and its possible mechanism, with a view to further elucidating the role of enkephalin in regulating the physiological function of the heart Awareness. Method: This lab is from four The rats were divided into control group (intravenous administration of equal volume of physiological saline), different concentrations of enkephalin 1 nM, and 10n. In the M and 50 nM groups, the left ventricle was placed retrograde with a carotid artery, and the LVSP, LVDP, + dP/ dtmax,-dP/ dtm were observed before and after administration. The changes of ax and HR were further selected by the combination of UFP-101 and enkephalin. To observe the change of the above-mentioned index.2. Immunofluorescence chemical calibration of the endorphin and its receptor: the distribution of the enkephalin and its receptor in the myocardium of the rat was observed by the aid of tissue immunofluorescence; and the use of Confcal in the myocardial cells of the rats with acute separation was used. The distribution of the endorphin receptor was observed.3. The L-type calcium channel, the inward rectifier potassium channel and the transient outward potassium channel of the cardiac myocyte were studied by using the technique of patch clamp, and the effect of the endorphin on the function of the ion channels was observed. The effect of enkephalin on the activity of PKA in the cardiac muscle cells was described.4. The effect of the enkephalin on the activity of the PKA in the cardiac muscle cells: the cardiac myocyte protein treated by different concentration of OFQ was extracted, and the activation range was determined by the PKA activity analysis kit. degree, in order to The possible molecular mechanism of enkephalin was discussed. Results:1. The VSP, LVDP, + dP/ dtmax,-dP/ dtmax and HR were significantly inhibited by the intravenous administration of enkephalin (P0.05). UFP-101, an antagonist of endorphin, can block this effect. The results of the experiment of the patch clamp showed that the enkephalin had an obvious inhibitory effect on the L-type calcium current in the cardiac muscle cells, but there was no significant dose-dependence, and the effect of 1 nM of the enkephalin was the most obvious (the inhibition rate was the inhibition rate). 22.7%, P 0.05), UFP-101 can block this inhibition. The enkephalin does not have a significant effect on the inward rectifier potassium current and the transient outward potassium current. The activity of PKA in the cardiac muscle cells showed that the PKA had no significant effect on the PKA of the cardiomyocytes. Conclusion: The effect of the enkephalin on the left ventricular contraction and the diastolic function of the rat heart is obviously inhibited by the increase of the activity of A. Conclusion: The effect of the enkephalin on the left ventricular contraction and the diastolic function of the rat heart is obviously inhibited. The effect of enkephalin on the activity of the L-type calcium current in the cardiac muscle cell may be caused by the effect of the enkephalin-enkephalin receptor. The further study shows that the PKA activity of the isolated enkephalin in the cardiac muscle cells is not There is an effect suggesting that this effect of enkephalin is not mediated by the effect of the cAMP-PKA pathway.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R33

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相關(guān)期刊論文 前1條

1 孟美金,Ting Wee Lee,Michael George Ricos,吳新民,Lee Tat Leang,Tachibana Shinro;鞘內(nèi)注射孤啡肽對(duì)不同小鼠所致痛覺超敏的比較[J];中華麻醉學(xué)雜志;2003年09期

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本文編號(hào)：2448569