本文關鍵詞： 阿里阿德涅同族體2 泛素結合酶E2Q1 腦損傷 中樞神經(jīng)系統(tǒng) 凋亡　出處：《南通大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的:研究Triad1與UBE2Q1在大鼠創(chuàng)傷性腦損傷中的表達變化,細胞定位,二者之間的相互作用以及在大鼠創(chuàng)傷性腦損傷后神經(jīng)修復中的意義,為創(chuàng)傷性腦損傷的臨床治療尋找新的途徑。方法:建立SD大鼠腦外傷模型,分別運用Western blot、RT-PCR、免疫熒光雙染等方法觀察腦損傷及炎癥反應過程中Triad1與UBE2Q1蛋白質(zhì)表達的時空變化和細胞定位。在細胞水平通過H2O2誘導Pc12細胞損傷反應,聯(lián)合運用基因克隆技術,通過干擾或過表達Triad1與UBE2Q1的表達來觀察Triad1與UBE2Q1及其相互作用與神經(jīng)元細胞損傷凋亡反應之間的關系。進一步通過細胞流式檢測Triad1與UBE2Q1及其相互作用與神經(jīng)元細胞損傷凋亡的關系。結果:在大鼠腦外傷模型中,我們發(fā)現(xiàn)Triad1表達水平增加,Western Blot及RT-PCR表明Triad1表達隨著時間點延長逐漸增高,3~7天到達高峰,后又逐漸降低,而UBE2Q1的表達水平與Triad1相反,5天表達水平較其他時間點降低;免疫熒光顯示在受損傷的大腦皮層中周圍,Triad1與UBE2Q1主要表達在神經(jīng)元中,星形膠質(zhì)細胞中較少,其陽性信號較正常組明顯增強;免疫熒光雙標證實Triad1與UBE2Q1共定位,同時通過免疫共沉淀從組織水平及細胞水平進一步證實Triad1與UBE2Q1存在相互作用,在腦損傷5天后的大腦皮層中和PC12細胞中,免疫熒光雙標試驗證實Triad1與UBE2Q1與活化的caspase-3、TUNEL共定位。同時活化的caspase-3、凋亡相關因子p53,Bax等表達水平增加,通過干擾或過表達Triad1與UBE2Q1,通過細胞流式方法進一步顯示在過表達Triad1與UBE2Q1后,細胞凋亡水平較干擾組明顯增加,并且依賴于p53的表達。結論:1創(chuàng)傷性腦損傷誘導Triad1與UBE2Q1的表達變化,且二者存在相作用的關系;2在創(chuàng)傷性腦損傷后,Triad1與UBE2Q1的相互作用及其表達變化促進了神經(jīng)元的凋亡;3細胞水平的研究結果進一步證實Triad1與UBE2Q1之間的相互作用及促進神經(jīng)元凋亡并依賴于p53的表達;4進一步明確了Triad1是E3泛素連接酶,具有泛素化降解的作用。
[Abstract]:Objective: to study the expression and localization of Triad1 and UBE2Q1 in traumatic brain injury (TBI) in rats, the interaction between them and their significance in nerve repair after traumatic brain injury (TBI) in rats. To find a new way for the clinical treatment of traumatic brain injury methods: to establish a rat model of traumatic brain injury. The temporal and spatial changes and cellular localization of Triad1 and UBE2Q1 protein expression during brain injury and inflammatory reaction were observed by Western blotr RT-PCR and immunofluorescence double staining, respectively. At the cellular level, the expression of Triad1 and UBE2Q1 protein was induced by H 2O 2 in combination with gene cloning technique. By interfering or overexpressing the expression of Triad1 and UBE2Q1, the relationship between Triad1 and UBE2Q1 and their interaction with neuronal cell injury and apoptosis was observed. Furthermore, the relationship between Triad1 and UBE2Q1 and their interaction with neuronal cells was detected by flow cytometry. Relationship between injury and apoptosis. Results: in the rat model of brain injury, We found that the expression level of Triad1 increased. Western Blot and RT-PCR showed that the expression of Triad1 increased gradually with the extension of time point and reached its peak at 3d, then decreased gradually. The expression level of UBE2Q1 was lower than that of Triad1 at 5 days as opposed to that of Triad1. Immunofluorescence showed that triad1 and UBE2Q1 were mainly expressed in neurons and astrocytes were less in injured cerebral cortex, and their positive signals were significantly higher than those in normal controls. Immunofluorescence double labeling confirmed that Triad1 and UBE2Q1 were co-localized. At the same time, the interaction between Triad1 and UBE2Q1 was further confirmed by co-immunoprecipitation at the tissue and cell levels, and in the cerebral cortex and PC12 cells 5 days after brain injury. Immunofluorescence double labeling assay confirmed that Triad1 and UBE2Q1 were co-located with activated caspase-3, and the expression of caspase-3 and apoptosis-related factor p53 + Bax were increased. By interfering or overexpressing Triad1 and UBE2Q1, the expression levels of Triad1 and UBE2Q1 were further demonstrated by cell flow cytometry. The level of apoptosis was significantly higher than that of interference group, and was dependent on the expression of p53. Conclusion the changes of Triad1 and UBE2Q1 expression induced by traumatic brain injury at 1: 1 were observed. The interaction between triad1 and UBE2Q1 and its expression after traumatic brain injury have promoted the apoptosis of neurons. The results further confirm the interaction between Triad1 and UBE2Q1 and promote the development of neuronal apoptosis. Apoptosis of neurons and dependence on p53 expression further confirmed that Triad1 is an E3 ubiquitin ligase. It has the function of ubiquitin degradation.
【學位授予單位】：南通大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R651.15
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本文編號：1553325