本文選題：缺血后適應(yīng) + 缺血/再灌注損傷�。� 參考：《昆明醫(yī)科大學(xué)》2016年博士論文

【摘要】：目的：本研究旨在通過(guò)microRNA芯片技術(shù)探討microRNAs在缺血／再灌注損傷及缺血后適應(yīng)后小鼠腦組織中的表達(dá)變化,篩選出差異表達(dá)的microRNAs,進(jìn)而進(jìn)一步在體內(nèi)及體外模型進(jìn)行驗(yàn)證,深入研究和探討miRNA參與腦缺血后適應(yīng)的神經(jīng)保護(hù)作用及其可能涉及的調(diào)控機(jī)制。方法：實(shí)驗(yàn)分為三部分,第一部分小鼠腦缺血后適應(yīng)microRNA表達(dá)變化的研究：在小鼠腦缺血/再灌注損傷的模型基礎(chǔ)上,通過(guò)microRNAs芯片篩查技術(shù)篩選出后適應(yīng)后大腦皮層及海馬組織內(nèi)差異性表達(dá)的microRNAs,采用qRT-PCR技術(shù)對(duì)差異表達(dá)明顯的miRNAs進(jìn)行驗(yàn)證,并利用信息學(xué)軟件進(jìn)行靶基因分析及篩選。第二部分：對(duì)差異表達(dá)變化明顯的miRNA-124進(jìn)行進(jìn)一步研究,探討miRNA-124參與腦缺血后適應(yīng)對(duì)小鼠缺血/再灌注損傷的神經(jīng)保護(hù)作用的機(jī)制：采用小鼠側(cè)腦室給藥的方案,給予miRNA-124激動(dòng)劑(agomir)及抑制劑(antagomir)處理,通過(guò)TTC染色法、TUNEL法、NeuN染色、Western blot、 qRT-PCR方法比較抑制及過(guò)表達(dá)miRNA-124后,腦梗死面積、細(xì)胞凋亡及存活情況、對(duì)PI3K/Akt2信號(hào)通路的影響、抗凋亡蛋白Bcl-2及凋亡相關(guān)蛋白Bax. Caspases 3的表達(dá)情況。第三部分miRNA-124調(diào)節(jié)PI3K/Akt2信號(hào)通路,減輕缺血再灌注損傷細(xì)胞凋亡的機(jī)制研究：采用PC12細(xì)胞體外實(shí)驗(yàn),氧糖剝奪法模擬缺血/再灌注損傷模型,進(jìn)一步驗(yàn)證miRNA-124通過(guò)調(diào)控PI3K/Akt2信號(hào)通路對(duì)抗缺血/再灌注損傷：建立PC12細(xì)胞氧糖剝奪/再灌注模型,niRNA-124agomir及antagomir進(jìn)行細(xì)胞預(yù)處理,抑制及過(guò)表達(dá)miRNA-124,采用超氧化物陰離子探針、CCK8法、Annexin V FITC/PI流式雙染、免疫熒光染色、Western blot、qRT-PCR等方法比較PC12細(xì)胞在缺血/灌注損傷后細(xì)胞缺氧情況、細(xì)胞活力及凋亡情況、PIK3/Akt2號(hào)通路、抗凋亡蛋白Bcl-2及凋亡相關(guān)蛋白Bax, Caspases 3的表達(dá)情況。結(jié)果：①microRNAs芯片篩查結(jié)果顯示：與腦缺血/再灌注組相比,后適應(yīng)處理后,小鼠皮層及海馬區(qū)多種miRNAs表達(dá)水平發(fā)生顯著變化,部分miRNA出現(xiàn)相同的變化,后適應(yīng)下調(diào)了miRNA-1,let-7和miRNA-124的表達(dá),上調(diào)了miRNA-19a的表達(dá)(p值均0.05)。通過(guò)qRT-PCI進(jìn)一步驗(yàn)證了miRNAs的表達(dá)變化(p0.05)。②體內(nèi)實(shí)驗(yàn)結(jié)果顯示：抑制miRNA-124的表達(dá)后協(xié)同了后適應(yīng)對(duì)缺血/再灌注損傷的腦保護(hù)作用,在后適應(yīng)基礎(chǔ)上進(jìn)一步縮小了腦梗死體積、保護(hù)了神經(jīng)細(xì)胞、減少了細(xì)胞凋亡,增強(qiáng)了PI3K/Akt2信號(hào)通路表達(dá),抗凋亡蛋白Bcl-2表達(dá)上調(diào)、凋亡相關(guān)蛋白Bax、Caspases3表達(dá)下調(diào)(P值均0.05),過(guò)表達(dá)miRNA-124后抵消了后適應(yīng)的腦保護(hù)效果。③體外實(shí)驗(yàn)結(jié)果顯示：抑制PC12細(xì)胞中miRNA-124的表達(dá)后PI3K/Akt2信號(hào)通路表達(dá)增強(qiáng),有效減少氧糖剝奪/再灌注導(dǎo)致的PC12細(xì)胞凋亡、增強(qiáng)細(xì)胞活力(P值均0.05),抗凋亡蛋白Bcl-2表達(dá)上調(diào)、凋亡相關(guān)蛋白Bax、Caspases3表達(dá)下調(diào)(P值均0.05),而過(guò)表達(dá)miRNA-124后加重了細(xì)胞缺血/再灌注損傷。結(jié)論：缺血后適應(yīng)通過(guò)調(diào)控皮層及海馬腦組織中miRNAs的表達(dá)水平對(duì)抗腦缺血/再灌注損傷。在小鼠I/R模型中,miRNA-1、let-7、miRNA19a和miRNA-124可能是與后適應(yīng)保護(hù)機(jī)制相關(guān)的miRNAs。miRNA-124參與了腦缺血/再灌注損傷病理生理過(guò)程,并通過(guò)靶向負(fù)性調(diào)控PI3K/Akt2通路參缺血后適應(yīng)在小鼠腦缺血/再灌注損傷中的抗凋亡作用。
[Abstract]:Objective: the purpose of this study was to investigate the changes in the expression of microRNAs in the cerebral tissue of mice after ischemia / reperfusion injury and post ischemic adaptation by microRNA chip technology, and to screen out the differential expression of microRNAs, and further verify in vivo and in vitro models, and further study and explore the neuroprotection of miRNA in the adaptation to cerebral ischemia. The effect and its possible regulatory mechanism. Methods: the experiment is divided into three parts. The first part is the study of the changes in the expression of microRNA after cerebral ischemia in mice: on the basis of the model of cerebral ischemia / reperfusion injury in mice, the differential expression of micro in the large cerebral cortex and hippocampus after adaptation is screened by microRNAs chip screening technique. RNAs, using qRT-PCR technology to verify the distinctly expressed miRNAs, and use the information software to carry out the target gene analysis and screening. The second part: to further study the miRNA-124 with distinct differential expression change, and explore the mechanism of miRNA-124 involved in the neuroprotective effect of miRNA-124 on the injury of blood / reperfusion injury in mice after cerebral ischemia Using the scheme of the mouse lateral ventricle administration, the miRNA-124 agonist (agomir) and the inhibitor (antagomir) were treated with TTC staining, TUNEL, NeuN staining, Western blot, and qRT-PCR methods to suppress and overexpress miRNA-124, the area of cerebral infarction, the cell withering and survival, the effect on the PI3K/Akt2 signaling pathway, and the anti apoptotic protein B The expression of Cl-2 and apoptosis related protein Bax. Caspases 3. Third part miRNA-124 regulates the PI3K/Akt2 signaling pathway to reduce the mechanism of apoptosis induced by ischemia-reperfusion injury: in vitro experiment of PC12 cells, oxygen glucose deprivation is used to simulate ischemia / reperfusion injury model, and one step is to verify that miRNA-124 is regulated by the PI3K/Akt2 signaling pathway. Against ischemia / reperfusion injury: PC12 cell oxygen glucose deprivation / reperfusion model, niRNA-124agomir and antagomir cell preconditioning, inhibition and overexpression of miRNA-124, using superoxide anion probe, CCK8, Annexin V FITC/PI flow double staining, immunofluorescence staining, Western blot, qRT-PCR and other methods to compare PC12 cells in ischemia / The condition of cell hypoxia, cell vitality and apoptosis, PIK3/Akt2 pathway, anti apoptotic protein Bcl-2 and apoptosis related protein Bax, Caspases 3 expression. Results: 1. The results of microRNAs chip screening showed that after the cerebral ischemia / reperfusion group, after adaptation, there were a variety of miRNAs expression water in the cortex and hippocampus of mice. There was a significant change in the level of miRNA, and the expression of miRNA-1, let-7 and miRNA-124 was down regulated, and the expression of miRNA-19a was up-regulated (P value was 0.05). The expression of miRNAs was further verified by qRT-PCI (P0.05). The cerebral protective effect of perfusion injury further narrowed the volume of cerebral infarction on the basis of post adaptation, protected the nerve cells, reduced the apoptosis, enhanced the expression of PI3K/Akt2 signaling pathway, increased the expression of anti apoptotic protein Bcl-2, Bax, Caspases3 expression (all P 0.05), and counteracted the postadaptation after overexpression of miRNA-124. The results of brain protection in vitro showed that the expression of PI3K/Akt2 signaling pathway was enhanced after the inhibition of miRNA-124 expression in PC12 cells, effectively reducing the apoptosis of PC12 cells induced by oxygen deprivation / reperfusion, enhanced cell viability (P value 0.05), up regulation of anti apoptotic protein Bcl-2 expression, and down regulation of apoptosis related protein Bax, Caspases3 expression (P value of 0). 5), after overexpression of miRNA-124, it aggravates cell ischemia / reperfusion injury. Conclusion: adaptation to ischemic / reperfusion injury by regulating the expression level of miRNAs in the cortex and hippocampal brain tissue after ischemia. In the mouse I/R model, miRNA-1, let-7, miRNA19a and miRNA-124 may be associated with the postadaptation mechanism of miRNAs.miRNA-124. The pathophysiological process of cerebral ischemia / reperfusion injury and the anti apoptosis effect of PI3K/Akt2 pathway on cerebral ischemia / reperfusion injury in mice are regulated by target negative regulation.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R743.3

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相關(guān)期刊論文 前1條

1 王路喬;程曉曙;黃茶花;黃波;梁茜;;Rapamycin Protects Cardiomyocytes against Anoxia/Reoxygenation Injury by Inducing Autophagy through the PI3k/Akt Pathway[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2015年01期

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本文編號(hào)：1989174