【作者】 张夔鸣；

【导师】 廖志钢；

【作者基本信息】 四川大学 ， 法医学， 2004， 硕士

【摘要】 研究背景 缺氧脑损伤(Hypoxic Brain Injury,HBI)是临床常见的病理现象,也是法医实践中各种原因所致窒息情况下最常发生的病变以及主要的死亡原因之一。中枢神经系统对缺氧敏感,脑组织耗氧量大、代谢率高,组织氧和ATP储备少;脑以有氧代谢为主,几乎没有无氧代谢能力,对缺氧耐受差,缺氧对其结构和功能影响最早且严重。 脑缺氧时相继发生神经元能量供求失衡,膜内外离子梯度消失,无氧去极化和细胞死亡。缺氧所致的神经元损害机制一般认为有坏死和凋亡两种细胞死亡形式。坏死对缺氧损伤反应迅速,在损伤后很快出现;凋亡是由基因控制的细胞主动死亡过程,需要新的蛋白质合成,以DNA的早期降解为特征,表现为迟发性的神经元死亡。缺氧后线粒体损伤,细胞发生能量缺失和代谢功能障碍;并介导凋亡调节信号传递,是缺氧后神经元损害的一个中心环节。线粒体膜通透性改变,释放细胞色素C(cytochrome c,Cyt C)活化特定的Caspase蛋白酶,导致细胞凋亡或使呼吸链电子传递中断促使细胞发生坏死。Bcl-2蛋白定位于线粒体膜上,对正常细胞的自身稳定性起重要作用,调控细胞色素C释放,抑制神经元凋亡。 Cyt C、Bcl-2在外伤性、缺血—缺氧性、中毒性脑损伤后均会出现神经元中表达变化,在不同模型中,由于存在许多非缺氧影响因素,表达变化的规律也不尽相同。缺氧后,脑的不同部位神经元CytC、Bcl-2表达随损伤后不同时间亦呈现一定的变化规律。目前单纯窒息缺氧所致神更多还原

【Abstract】 BackgroundHypoxic brain injury(HBI) is known to a familiar clinic pathological phenomena , and one of the mainly cause of death that induced by sorts of anoxic damage in forensic cases. The central nervous system(CNS) is very sensitive to hypoxia, brain tissue have a high oxygen-consumption and metabolism rate, moreover, the reserve of tissue oxygen and ATP is limited. The energy supply of nervous tissue depend on aerobic metabolism mostly, with little capability of anaerobic metabolism and poor tolerance to anoxia. The lesion often occurs in early stage when hypoxia happened , which influenced brain’s structure and function extensively .After hypoxia injury, many changes emerge in neurons include losing the balance of energy supply and consumption, the ion gradient between inner and outer membrane of cell disappeared, anaerobic depolarization and cell death. There are two distinct ways about neurons death after hypoxia: necrosis and apoptosis. Necrosis is the pathological response process of inflammation, which occurs rapidly when cells are exposed to a anoxic insult. Apoptosis is characterized by DNA degradation and active death process which be control by gene, this process need new proteins synthesized, the neurons often occur delayed death. Hypoxic insult to neuron’s mitochondrion induce deficiency ofenergy and dysfunction of metabolism, mediate the transfer of apoptosis signal, the damage to mitochondrion is a key step in neuron injury mechanism. Accompanied alters of the mitochondrial permeability, released cytochrome C from mitochondrion active Caspase proteinase which lead to neuron apoptosis, or to promote neuron’s necrosis by interrupting the electronic transport of respiratory chain. Bcl-2 protein locates on the mitochondrial membrane, undergoes a important role to keep the stability of cell, and inhibits neuron’s apoptosis by regulating and modulating the releasing of cytochrome C and other pro-apoptosis proteins.Many study have observed the expressing variety of cytochrome C and Bcl-2 in brain after trauma, ischemia-hypoxia and intoxication. In different models, expressing rule of cytochrome C and Bcl-2 is different because of many non-hypoxia factors involved. After hypoxia injury, cytochrome C and Bcl-2 expressing demonstrate some regularly variety related with time course of post-hypoxia in different brain regions. In present, there are few report about cytochrome C and Bcl-2 expression under only oxygen deprived brain injury, there is no research reported about the significance of the expressing in brain which be concerned with forensic applications after hypoxic brain injury induced by mechanical asphyxia. ObjectiveStudy the expression changes of cytochrome C and Bcl-2 following hypoxic brain injury on the basis of duplicate acute continual hypoxia model, to discuss the molecular pathological mechanism of post-hypoxia neuron injury; to provide theoretical basis for estimation of time since brain hypoxia in forensic and clinic practice. MethodsFifty-five Spragwe-Dawley rats were randomly divided into normal control, sham hypoxia control and hypoxic injury groups. The experimental rats were subjected to continual hypoxic brain injury by a 1000ml airproofed glass bottle with anoxic condition. According to the different survival time ,the hypoxia group were then subdivided into post-hypoxia instant, 30min, lh, 3h, 6h, 12h, 24h, 48h, and 72h groups. The changes and absence of neurons in different brain regions was studied by Nissl’s Toluidine Blue Staining. The expressing of cytochrome C and Bcl-2 in brain was studied by immunohistochemistry (IHC). Result(1) In the groups of normal control and sham hypoxia control rats, the expressing level of cytochrome C and Bcl-2 is low. (2) The expressing of cytochrome C and Bcl-2 was upreguiated following hypoxia. The expressing of cytochrome C increasing at at 3h, reaching peak at 12-24h, and maintaining high expression level at 48h and falling to normal at 72h after brain hypoxia. Whereas, Bcl-2 was increasing at lh, reach peak at 6h and falling to lowest level at 72h after hypoxia. (3) The expressing of Cyt C and Bcl-2 are both upreguiated in cerebral cortex, hippocampus, and thalamencephalon. The change regularity of cytochrome C and Bcl-2 shows obvious relationship with the survival time after hypoxia. Furthermore, The expressing of cytochrome C and Bcl-2 change pattern showed no difference in the three brain areas. (4) Nissl body in neuron’s cytoplasm lessen or disappear in various brain regions, that shows hypoxia injury lead to neurons absence. ConclusionHypoxia brain injury could induce the expressing of cytochrome C and Bcl-2 in various brain regions and the changes of cytochrome C and Bcl-2 showed time sequence regularity. Therefore, cytochrome C and Bcl-2 could be applied to estimate the time since hypoxia as an objective indication.The time regulation of cytochrome C and Bcl-2 expressing, the extent of change and abnormal distributing of cytochrome C and Bcl-2 could be applied to estimate the survival situation and damage degree, and to find potential protective measures.更多还原