受体相互作用蛋白3及经典程序性坏死信号通路在脑缺血再灌注损伤中的作用研究

发布时间：2018-04-16 23:18

本文选题：程序性坏死 + 受体相互作用蛋白　；参考：《华中科技大学》2015年博士论文

【摘要】：[背景]细胞死亡涉及到生命的多个环节,正面作用有：胚胎的发育,机体细胞的“新陈代谢”,反面左右有：炎症、肿瘤等。既往认为细胞死亡中除细胞凋亡是基因预存的主动死亡模式,是由细胞产生各种信号分子伴随能量消耗最终导致细胞死亡之外,其他的细胞死亡都是被动的不可逆的细胞死亡。2005年Degterev等提出一种新的细胞死亡方式：细胞程序性坏死。细胞的这种死亡方式具有坏死样结构特点,细胞膜完整性破坏,周围组织炎症反应,同时该过程受细胞内一系列信号分子调控。脑缺血所致脑损伤是我国神经科致死、致残的主要病因之一。如果能通过深入探索程序性坏死信号通路的生物化学机制,更进一步研究程序性坏死信号通路在相关疾病中的参与程度与作用,不但利于对该信号通路的认知,且对神经内外科相关神经缺血再灌注损伤相关疾病的治疗药物、治疗靶向、治疗方法起到拓宽、确立的重要作用。[目的]本实验研究体外培养神经元细胞在缺血再灌注损伤后RIP3的变化规律。本实验受临床上脑梗死后出现缺血半暗带启发,研究并探讨程序性坏死在半暗带区和梗死区的分布,进一步揭示程序性坏死在脑缺血再灌注损伤中的作用机制。[方法]原代神经元培养,神经元细胞荧光鉴定,RIP3荧光鉴定,传代细胞HT22细胞培养及冻存,细胞造缺氧模型,动物造缺氧再灌注模型,PCR技术,WesternB1otting技术,MTT技术,鼠脑TTC染色[结果]RIP3表达在缺血再灌注在体和体外模型中都有显著变化。在本实验所设之间点中,在缺血再灌注损伤后12小时RIP3表达达到高峰。小鼠海马来源细胞系HT22与大鼠皮层神经元造模后PCR结果趋势一致。动物造模后梗死区及半暗带区Western Blotting趋势一致,与细胞PCR趋势一致。在体实验中,梗死区较半暗带区,RIP3表达波动显著。缺氧时间1小时NEC-1实验组细胞存活率较对照组升高不明显,而缺氧时间为4小时NEC-1实验组细胞存活率较对照组显著升高。[结论]脑缺血再灌注模型中,缺血区和半暗带区由于损伤程度不同,程序性坏死参与度不同,梗死区程序性坏死更活跃。[创新与不足]本实验受临床上脑梗死后出现缺血半暗带启发,探讨程序性坏死在半暗带区和梗死区的分布,进一步揭示程序性坏死在脑缺血再灌注损伤中的作用机制。动物模型中干预后组织细胞的坏死变化是否能或只能归因于程序性坏死成为疑问,这使结果分析变得困难,一些实验结果不能用己知道理论解释。
[Abstract]:Background: cell death is related to many aspects of life. The positive effects are: embryo development, cell metabolism, inflammation, tumor and so on.It has been thought that in cell death, except apoptosis is the active death mode of gene pre-storage, it is caused by various signal molecules produced by the cell and energy consumption, which leads to cell death.Other cell death is passive irreversible cell death. In 2005 Degterev et al proposed a new cell death mode: programmed cell necrosis.The cell death mode has the characteristics of necrotic structure, cell membrane integrity destruction, inflammation of surrounding tissues, and the process is regulated by a series of signaling molecules in the cell.Brain injury caused by cerebral ischemia is one of the main causes of death and disability in neurology in China.If we can further study the degree and function of the process necrosis signal pathway in the related diseases by further exploring the biochemical mechanism of the process necrosis signal pathway, it is not only beneficial to the cognition of the signal pathway,It also plays an important role in the treatment of nerve ischemia-reperfusion injury related diseases.[objective] to study the changes of RIP3 in cultured neuron cells after ischemia reperfusion injury in vitro.The present study was inspired by the occurrence of ischemic penumbra after cerebral infarction. The distribution of programmed necrosis in the penumbra and infarct area was studied, and the mechanism of programmed necrosis in cerebral ischemia-reperfusion injury was revealed.[methods] Primary culture of neurons, fluorescence identification of neuron cells and fluorescence identification of RIP3, culture and freezing of passage HT22 cells, establishment of anoxic model of cells, and establishment of hypoxia reperfusion model in animals were studied by Western B1otting technique.TTC staining [results] the expression of RIP3 was significantly changed in both in vivo and in vitro models.The expression of RIP3 reached its peak 12 hours after ischemia reperfusion injury.Mouse hippocampal derived cell line HT22 and rat cortical neurons showed the same trend of PCR results.The trend of Western Blotting in infarct and penumbra was the same as that in cell PCR.In vivo, RIP3 expression fluctuated significantly in infarcted area than in penumbra zone.The cell survival rate of NEC-1 group was not significantly higher than that of control group, but the cell survival rate of NEC-1 group was significantly higher than that of control group when hypoxia time was 4 hours.[conclusion] in the model of cerebral ischemia-reperfusion, the ischemic and penumbra regions are more active because of the different degree of injury and the participation degree of programmed necrosis.[innovation and deficiency] this experiment is inspired by the occurrence of ischemic penumbra after clinical cerebral infarction. The distribution of programmed necrosis in penumbra and infarct area is discussed, and the mechanism of procedural necrosis in cerebral ischemia-reperfusion injury is further revealed.Whether or not the necrotic changes of tissue cells after intervention in animal models can or can only be attributed to programmed necrosis is questionable, which makes the analysis of results difficult, and some experimental results cannot be explained by known theories.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R743.3

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