Triad1与UBE2Q1的相互作用在大鼠创伤性脑损伤中的作用的研究

发布时间：2018-03-01 19:40

  本文关键词： 阿里阿德涅同族体2 泛素结合酶E2Q1 脑损伤 中枢神经系统 凋亡　出处：《南通大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的:研究Triad1与UBE2Q1在大鼠创伤性脑损伤中的表达变化,细胞定位,二者之间的相互作用以及在大鼠创伤性脑损伤后神经修复中的意义,为创伤性脑损伤的临床治疗寻找新的途径。方法:建立SD大鼠脑外伤模型,分别运用Western blot、RT-PCR、免疫荧光双染等方法观察脑损伤及炎症反应过程中Triad1与UBE2Q1蛋白质表达的时空变化和细胞定位。在细胞水平通过H2O2诱导Pc12细胞损伤反应,联合运用基因克隆技术,通过干扰或过表达Triad1与UBE2Q1的表达来观察Triad1与UBE2Q1及其相互作用与神经元细胞损伤凋亡反应之间的关系。进一步通过细胞流式检测Triad1与UBE2Q1及其相互作用与神经元细胞损伤凋亡的关系。结果:在大鼠脑外伤模型中,我们发现Triad1表达水平增加,Western Blot及RT-PCR表明Triad1表达随着时间点延长逐渐增高,3~7天到达高峰,后又逐渐降低,而UBE2Q1的表达水平与Triad1相反,5天表达水平较其他时间点降低;免疫荧光显示在受损伤的大脑皮层中周围,Triad1与UBE2Q1主要表达在神经元中,星形胶质细胞中较少,其阳性信号较正常组明显增强;免疫荧光双标证实Triad1与UBE2Q1共定位,同时通过免疫共沉淀从组织水平及细胞水平进一步证实Triad1与UBE2Q1存在相互作用,在脑损伤5天后的大脑皮层中和PC12细胞中,免疫荧光双标试验证实Triad1与UBE2Q1与活化的caspase-3、TUNEL共定位。同时活化的caspase-3、凋亡相关因子p53,Bax等表达水平增加,通过干扰或过表达Triad1与UBE2Q1,通过细胞流式方法进一步显示在过表达Triad1与UBE2Q1后,细胞凋亡水平较干扰组明显增加,并且依赖于p53的表达。结论:1创伤性脑损伤诱导Triad1与UBE2Q1的表达变化,且二者存在相作用的关系;2在创伤性脑损伤后,Triad1与UBE2Q1的相互作用及其表达变化促进了神经元的凋亡;3细胞水平的研究结果进一步证实Triad1与UBE2Q1之间的相互作用及促进神经元凋亡并依赖于p53的表达;4进一步明确了Triad1是E3泛素连接酶,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.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R651.15
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本文编号：1553325