【摘要】：烯醇化酶磷酸酶1(Enolase-phosphatase 1 ENOPH1)是甲硫氨酸補(bǔ)救途徑的重要蛋白分子,具有磷酸酶功能,屬于非典型烯醇化酶活性的雙功能酶,在調(diào)節(jié)氧化應(yīng)激反應(yīng)中發(fā)揮著重要作用。本次研究探究的是ENOPH1在急性缺血性腦卒中血腦屏障損傷中扮演的角色。前期研究發(fā)現(xiàn):局灶性腦缺血可誘導(dǎo)大鼠腦微血管組織中ENOPH1的mRNA和蛋白表達(dá)水平增高。本研究通過(guò)乳酸脫氫酶釋放法及TUNEL法檢測(cè)到3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴化物有所增加,提示缺血缺氧條件(oxygen-glucose deprivation,OGD)下,腦微血管內(nèi)皮細(xì)胞(bEND3 cells)內(nèi)ENOPH1的mRNA和蛋白表達(dá)水平上調(diào),同時(shí)伴隨著細(xì)胞凋亡增加。通過(guò)質(zhì)粒轉(zhuǎn)染法敲低ENOPH1和過(guò)表達(dá)ENOPH1蛋白水平可分別減少和增加OGD誘導(dǎo)下內(nèi)皮細(xì)胞凋亡,甚至,大幅度擴(kuò)大或減少氧化應(yīng)激反應(yīng)內(nèi)凋亡相關(guān)蛋白(如caspase-3,PARP,Bcl-2 and Bax)和內(nèi)質(zhì)網(wǎng)應(yīng)激蛋白(Ire-1,Calnexin,GRP78 and PERK)的表達(dá)水平。同樣發(fā)現(xiàn)OGD條件下還可誘導(dǎo)ENOPH1下游蛋白分子乙酸二羥丙烯醛加雙氧酶(acireductone dioxygenase 1,ADI1)表達(dá)水平上調(diào),并且OGD可促進(jìn)ENOPH1與ADI1之間的相互作用。但有趣的是,敲低ENOPH1并未參與OGD誘導(dǎo)下ADI1蛋白表達(dá)水平上調(diào),而只是調(diào)節(jié)ADI1從細(xì)胞核向細(xì)胞質(zhì)轉(zhuǎn)運(yùn)的過(guò)程。最后還發(fā)現(xiàn)敲低ENOPH1可顯著降低OGD誘導(dǎo)的內(nèi)皮細(xì)胞通透性增加。由此,本研究得出的結(jié)論是:ENOPH1的激活通過(guò)促進(jìn)氧化應(yīng)激反應(yīng),增加促凋亡相關(guān)蛋白的表達(dá),從而加重OGD誘導(dǎo)下內(nèi)皮細(xì)胞凋亡和血腦屏障損傷。因此,ENOPH1是治療急性缺血性腦卒中新的靶點(diǎn)。
[Abstract]:Enolase phosphatase 1 (Enolase-phosphatase 1 ENOPH1) is an important protein molecule of methionine remediation pathway. It has phosphatase function and belongs to a bifunctional enzyme with atypical enolase activity, which plays an important role in the regulation of oxidative stress reaction. This study explores the role of ENOPH1 in blood-brain barrier damage in acute ischemic stroke. Previous studies have shown that focal cerebral ischemia can induce increased expression of ENOPH1 mRNA and protein in rat brain microvascular tissue. In this study, lactate dehydrogenase release assay and TUNEL assay were used to detect the increase of 3- (4-dimethylthiazole-2) -2-diphenyl tetrazolium bromide, which suggested that the expression of mRNA and protein in cerebral microvascular endothelial cells (bEND3 cells) was up-regulated under ischemia and hypoxia (oxygen-glucose deprivation,OGD) conditions. At the same time, apoptosis was increased. Knockout and overexpression of ENOPH1 protein by plasmid transfection can decrease and increase the apoptosis of endothelial cells induced by OGD respectively. The expression of apoptosis-related proteins (such as caspase-3,PARP,Bcl-2 and Bax) and endoplasmic reticulum stress protein (Ire-1,Calnexin,GRP78 and PERK) in oxidative stress was significantly increased or decreased. It was also found that the expression of dihydroxyacrolein acetate (acireductone dioxygenase _ 1), a protein molecule in the downstream of ENOPH1, could be up-regulated by OGD, and that OGD could promote the interaction between ENOPH1 and ADI1. Interestingly, knockout ENOPH1 was not involved in the up-regulation of ADI1 protein expression induced by OGD, but only in the process of ADI1 transport from nucleus to cytoplasm. It was also found that knockout of ENOPH1 could significantly reduce the increase of endothelial cell permeability induced by OGD. It is concluded that the activation of ENOPH1 increases the expression of pro-apoptosis-related protein by promoting oxidative stress response, thereby exacerbating endothelial cell apoptosis and blood-brain barrier injury induced by OGD. Therefore, ENOPH1 is a new target for the treatment of acute ischemic stroke.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R743.3

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本文編號(hào)：2290703