本文選題：糖基化終末產(chǎn)物受體　切入點(diǎn)：蛛網(wǎng)膜下腔出血　出處：《南京大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：背景:蛛網(wǎng)膜下腔出血(subarachnoid hemorrhage,SAH)是高致死率和致殘率的中樞神經(jīng)系統(tǒng)血管性疾病。大量研究證實(shí)早期腦損傷(early brain injury,EBI)是造成SAH患者預(yù)后不良的主要原因。EBI的機(jī)制復(fù)雜,包括血腫機(jī)械損傷、腦灌注壓下降、血液成分毒性作用、腦水腫、氧化應(yīng)激、炎性反應(yīng)、神經(jīng)細(xì)胞凋亡壞死等。臨床治療中通過單一機(jī)制干預(yù)早期腦損傷無法取得滿意治療效果。因此尋找在SAH后可同時(shí)調(diào)控多種損傷機(jī)制的因子或因素顯得愈發(fā)重要。國際最新研究表明糖基化終末產(chǎn)物受體(receptor for advanced glycation end-product,RAGE)可同時(shí)介導(dǎo)炎性反應(yīng),調(diào)控細(xì)胞凋亡與自噬,參與神經(jīng)修復(fù)。同時(shí),RAGE的多種配體被證實(shí)參與SAH后腦損傷的病理生理過程。我們推測(cè)RAGE在SAH后早期腦損傷的發(fā)生發(fā)展中起到一定的作用。目前文獻(xiàn)中尚無關(guān)于RAGE在SAH后早期腦損傷中作用的報(bào)道。因此,本課題針對(duì)RAGE在SAH后的表達(dá)時(shí)程及其在早期腦損傷中的潛在作用機(jī)制進(jìn)行研究。方法:本課題分別建立SAH體內(nèi)及體外模型,首先運(yùn)用Western blotting、real-time PCR、免疫組織化學(xué)染色及免疫熒光技術(shù)在SAH后不同時(shí)間點(diǎn)觀察RAGE表達(dá)水平的變化及其細(xì)胞定位。然后根據(jù)體內(nèi)體外實(shí)驗(yàn)中RAGE的表達(dá)情況,使用其特異性抑制劑FPS-ZM1針對(duì)RAGE進(jìn)行下調(diào),運(yùn)用Western blotting、酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)、免疫組化及免疫熒光技術(shù)檢測(cè)核因子-κB(NF-κB)、TNF-α及IL-1β等炎性因子的表達(dá)變化及小膠質(zhì)細(xì)胞的活化水平變化來明確RAGE在SAH后炎性反應(yīng)中的作用及機(jī)制。然后,采用Flouro-JadeB、Nissl染色及流式細(xì)胞術(shù)分別從體內(nèi)體外模型中觀察下調(diào)RAGE后神經(jīng)細(xì)胞損傷情況。為明確RAGE在SAH后神經(jīng)損傷中的具體作用機(jī)制,我們進(jìn)一步采用Western blotting、免疫熒光技術(shù)檢測(cè)凋亡相關(guān)因子(caspase-3、Bcl-2、Bax)及自噬相關(guān)因子(LC3、beclin-1)的表達(dá)變化。最后,為明確下調(diào)RAGE對(duì)大鼠SAH后神經(jīng)功能的影響,我們檢測(cè)了大鼠腦組織含水量及神經(jīng)功能評(píng)分的變化。結(jié)果:體內(nèi)及體外實(shí)驗(yàn)結(jié)果均表明:RAGE在SAH后表達(dá)水平均較對(duì)照組明顯增高,同時(shí)RAGE的表達(dá)見于神經(jīng)元及小膠質(zhì)細(xì)胞中。SAH后抑制RAGE可顯著降低NF-κB及其下游炎性因子TNF-α和IL-1β的蛋白水平,并顯著抑制小膠質(zhì)細(xì)胞的活化。提示RAGE可能通過激活NF-κB及調(diào)控小膠質(zhì)細(xì)胞介導(dǎo)SAH后的炎性反應(yīng)。進(jìn)一步研究發(fā)現(xiàn),使用特異性抑制劑下調(diào)RAGE的表達(dá)后神經(jīng)細(xì)胞損傷加重,抑制RAGE引起神經(jīng)細(xì)胞凋亡增加,自噬減少。最后,體內(nèi)實(shí)驗(yàn)發(fā)現(xiàn)抑制RAGE可顯著減輕SAH后1天大鼠腦水腫,改善神經(jīng)功能,但不能改善SAH后3天大鼠腦組織水腫及神經(jīng)功能。結(jié)論:本課題通過體內(nèi)、體外實(shí)驗(yàn)證實(shí):SAH后RAGE的表達(dá)水平顯著增高,并在SAH后早期腦損傷中發(fā)揮"雙向作用",一方面通過NF-kB促進(jìn)炎性反應(yīng)引起腦損傷,另一方面通過調(diào)控凋亡與自噬減少神經(jīng)細(xì)胞死亡起到腦保護(hù)作用。本課題的發(fā)現(xiàn)有助于進(jìn)一步了解SAH后早期腦損傷的具體機(jī)制,也提示通過干預(yù)RAGE來治療SAH可能存在潛在的局限性。
[Abstract]:Background: subarachnoid hemorrhage (subarachnoid hemorrhage SAH) is a high mortality rate and disability rate of vascular diseases in central nervous system. Many studies have confirmed that early brain injury (early brain, injury, EBI) is the main reason causing mechanism of.EBI SAH patients with poor prognosis of the complex, including hematoma mechanical damage, cerebral perfusion pressure decreased blood components, toxicity, brain edema, oxidative stress, inflammatory reaction, apoptosis and necrosis. The clinical treatment by single intervention mechanism of early brain injury can not be achieved satisfactory results. So looking at SAH after injury and the regulation mechanism of various factors or factors become increasingly important. The latest research shows that the international sugar group end product receptor (receptor for advanced glycation end-product, RAGE) can mediate inflammatory reaction, cell apoptosis and autophagy, involved in nerve repair. At the same time, a variety of RAGE The ligand proved to be involved in the pathophysiological process of brain injury after SAH. We speculated that RAGE in early brain injury after SAH play an important role in the development of the literature. There is no report about RAGE in early brain injury after SAH effect. Therefore, the topic for the RAGE in the SAH after the expression of history and Study on the potential role in mechanisms of early brain injury. Methods: SAH in vivo and in vitro models are established in this paper, we first use the Western blotting, real-time PCR, immunohistochemical staining and immunofluorescence technique in SAH at different time after the observation of the change of the expression of RAGE and its cellular localization. Then according to the expression of RAGE in vivo and in vitro the use of its inhibitor FPS-ZM1 for RAGE reduction, using Western blotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and immunofluorescence detection of nuclear factor Sub - kappa B (NF- K B), activated expression of TNF- alpha and IL-1 beta and other inflammatory factors and microglia to clear level changes and mechanism of RAGE in inflammatory reaction after SAH. Then, using Flouro-JadeB, Nissl staining and observe respectively the neuronal damage after down-regulation of RAGE in vivo in vitro model of medium flow cytometry. To determine the RAGE in specific mechanism of nerve injury in SAH, we used Western blotting, immunofluorescence detection of apoptosis related factors (Caspase-3, Bcl-2, Bax) and autophagy related factors (LC3, beclin-1) expression changes. Finally, to clear the neurological effects of down-regulation of RAGE function after SAH in rats, we examined the changes of brain water content and neurological score. Results: in vivo and in vitro experimental results show that the expression of RAGE in SAH were higher than control group, while the expression of RAGE. 浜庣緇忓厓鍙?qiáng)灏忚兌璐ňl嗚優(yōu)涓，

本文編號(hào)：1650654