转录调节因子FoxO3a在高压氧致急、慢性氧中毒中的作用研究

发布时间：2018-01-06 22:41

本文关键词：转录调节因子FoxO3a在高压氧致急、慢性氧中毒中的作用研究　出处：《第二军医大学》2016年硕士论文　论文类型：学位论文

【摘要】：人们把超过一个大气压的压力叫做高气压,将机体置于高气压环境中,呼吸纯氧治疗疾病的方法称为高压氧(HBO)疗法。高压氧疗法已经被广泛地应用于潜水作业、潜艇脱险和临床相关疾病的治疗。在潜水作业后,呼吸高压氧进行减压,可使潜水过程中体内产生的惰性气体快速被置换出来,从而有效缩短减压时间,提高工作效率,保护潜水员的生命安全。潜艇失事后,潜艇艇员在紧急情况下,预防性吸入高压氧后再进行快速漂浮脱险,可以确保艇员生命安全,同时显著减轻减压病的病情及后遗症。在临床上,高压氧能够改善组织缺氧状况,促进损伤组织的修复和再生,对高血压脑出血、糖尿病足、脊髓损伤等具有一定的疗效。但是机体持续吸入氧分压过高的气体后,可能会出现毒性反应。当氧分压200kPa时,毒性反应会在较短时间内发生,并以中枢神经系统(CNS)功能紊乱为主要表现,称为中枢神经系统氧中毒(CNS-OT)或急性氧中毒(acute oxygen toxicity),其最剧烈、最典型的表现是类似于癫痫大发作样的全身强直性痉挛,即惊厥大发作,也称为“氧惊厥”。氧惊厥发生的同时,机体也会出现以肺出血为主要表现的急性肺损伤。当氧分压在60~200 kPa时,氧中毒损伤的主要靶器官是肺组织,主要表现为类似于支气管肺炎的症状,称为慢性氧中毒(chronic oxygen toxicity)。氧中毒的发生严重限制了高压氧的应用,深入研究氧中毒的发生发展过程及其发病机制,探索其致病的关键因素,对于有效预防氧中毒的发生具有非常重要的现实意义。近年的研究表明,转录调节因子FoxO3a在氧化应激中具有非常重要的作用。FoxO3a既能诱导细胞在氧化应激条件下凋亡,也能保护细胞在氧化应激条件下存活,且不同的细胞、组织在不同的氧化应激条件下,可能通过不同的机制发挥着多样的功能。PI3K-Akt-FoxO3a信号通路在氧化应激过程中发挥着重要的作用,被认为是FoxO3a调节氧化应激的一个重要信号通路。在PI3K/Akt/FoxO3a信号通路中,FoxO3a的苏氨酸残基32(Thr32)、丝氨酸残基253(Ser253)和丝氨酸残基315(Ser315)位点发生磷酸化后,可以介导FoxO3a由胞核转移到胞浆,转录活性受到抑制,下游相应的生物学效应也随之改变。当PI3K/Akt/FoxO3a途径受到抑制时,去磷酸化的FoxO3a大部分在细胞核聚集,激活下游靶基因的表达,进而调控相关的生物学功能。本课题旨在通过将小鼠经过高压氧暴露后观察其脑组织和肺组织的病理变化及相关氧化指标的变化,探索FoxO3a在急、慢性氧中毒的发生发展过程中是否发挥作用。同时探讨和确证FoxO3a在急、慢性氧中毒发生发展过程中的作用及其可能的机制,我们开展的研究和取得的结果主要有:第一部分转录调节因子FoxO3a在高压氧致急性氧中毒中的作用研究将小鼠进行了6 ATA,30 min的高压氧暴露,建立了急性氧中毒模型。通过观察FoxO3a基因敲除小鼠惊厥潜伏期和发作次数评估脑损伤情况,利用病理学切片和肺泡灌洗液中蛋白定量检测评估了肺组织的损伤情况,通过检测抗氧化酶活性、氧化产物指标等探索FoxO3a在其中的作用机制。小鼠高压氧暴露后,Western blot法检测大脑皮层和肺组织中FoxO3a蛋白表达情况,免疫组化定位FoxO3a,探索FoxO3a在氧化应激过程中可能的信号通路及机制。给予正常小鼠PI3K/AKT抑制剂LY294002阻断小鼠脑组织和肺组织的PI3K/Akt/FoxO3a通路,评估小鼠在高压氧暴露后脑组织和肺组织损伤情况。结果发现,FoxO3a基因敲除小鼠的惊厥潜伏期比未敲除小鼠显著地缩短,且30 min内惊厥的次数明显增多,FoxO3a基因敲除小鼠肺部总体损伤情况较未敲除小鼠严重;高压氧暴露结束后8 h,FoxO3a蛋白含量达到峰值,且蛋白发生了明显的入核;PI3K/AKT抑制剂LY294002抑制FoxO3a的主要信号通路后,FoxO3a明显被去磷酸化,从胞浆转位进入胞核中,脑损伤和肺组织损伤情况明显缓解。第二部分转录调节因子FoxO3a在高压氧致慢性氧中毒中的作用研究将小鼠进行了2.5 ATA,6 h的高压氧暴露,建立了慢性氧中毒模型。检测大脑皮层和肺组织中FoxO3a蛋白表达情况和胞浆、胞核的定位情况;PI3K/AKT抑制剂LY294002阻断PI3K/Akt/FoxO3a通路,评估小鼠在高压氧暴露后肺组织损伤情况。结果发现,高压氧暴露结束后8 h,FoxO3a蛋白含量达到峰值,且发生了明显的入核过程;PI3K/AKT抑制剂LY294002使FoxO3a发生去磷酸化,使其从胞浆转位进入胞核中,进而缓解肺组织损伤情况。研究结果表明:FoxO3a能够明显减轻高压氧引起的急、慢性氧中毒;高压氧暴露能够主动上调FoxO3a的蛋白水平和促使其入核来对抗氧中毒的发生;抑制PI3K/AKT信号通路使得FoxO3a发生入核,提高了FoxO3a的活性,脑和肺损伤情况明显缓解,进一步确证FoxO3a是进入胞核后发挥保护作用的。
[Abstract]:People have more than one atmospheric pressure is called high pressure, the body is placed in a high pressure environment, methods of treating a disease called breathing pure oxygen hyperbaric oxygen (HBO) therapy. Hyperbaric oxygen therapy has been widely used in the treatment of diving, submarine escape and clinical related diseases. In diving, breathing hyperbaric oxygen decompression, the inert gas produced in vivo in the process of rapid diving is replaced, it can effectively shorten the decompression time, improve work efficiency, protect the safety of life. The diver wreck submarine, the submarine crew in case of emergency, prevention of hyperbaric oxygen inhalation after rapid ascent, can ensure the life of sailors safety, also significantly reduced the decompression disease and sequelae. Clinically, hyperbaric oxygen can improve tissue hypoxia, promoting the repair and regeneration of damaged tissue, cerebral hemorrhage of high blood pressure, diabetes Foot, has a certain effect on spinal cord injury. But the continuous inhalation of oxygen gas pressure is too high, there may be a toxic reaction. When the oxygen pressure 200kPa, toxic reaction will occur in a relatively short period of time, and in the central nervous system (CNS) dysfunction as the main performance, said the central nervous poisoning oxygen system (CNS-OT) or acute oxygen poisoning (acute oxygen toxicity), the most intense, the most typical performance is similar to epileptic seizures like generalized tonic clonic seizures, namely convulsion, also known as the "convulsion convulsion". At the same time, the body will also appear acute lung injury with pulmonary hemorrhage as the main performance. When the oxygen partial pressure in the 60~200 kPa, the main target organ oxygen poisoning damage is mainly for lung tissue, similar to the symptoms of pneumonia, chronic oxygen poisoning "(chronic oxygen toxicity). The occurrence of serious restricted high toxic oxygen Application of oxygen pressure, in-depth study of the occurrence and development process and mechanism of oxygen poisoning, explore the key factors of the disease, has very important practical significance to effectively prevent occurrence of oxygen poisoning. Recent studies show that the transcription factor FoxO3a in oxidative stress has a very important role in.FoxO3a can induce apoptosis in under oxidative stress, can also protect the cell survival under oxidative stress conditions, and different tissue cells, oxidative stress in different conditions, may use different mechanisms to play the function of.PI3K-Akt-FoxO3a signaling pathway diversity plays an important role in the process of oxidative stress, is considered to be an important signaling pathway regulating FoxO3a oxidative stress. In the PI3K/Akt/FoxO3a signaling pathway, FoxO3a threonine residue 32 (Thr32), serine residue 253 (Ser253) and serine residues 315 (Ser315) Occurrence of phosphorylation sites, mediated FoxO3a transfer from nucleus to cytoplasm, transcriptional activity is inhibited, the downstream biological effects corresponding change. When the PI3K/Akt/FoxO3a pathway was inhibited, to most phosphorylated FoxO3a aggregation in the nucleus and activate the expression of downstream target genes, and regulate the biological functions related to this. The aims of the changes of mice after hyperbaric oxygen exposure to observe the pathological changes of the brain tissue and lung tissue and oxidation index, explore FoxO3a in acute, chronic oxygen poisoning is to play a role in the process of development. At the same time to investigate and confirm FoxO3a in acute, chronic oxygen poisoning occurred in the process of development and its possible role we carry out the research of mechanism, and the main achievements are as follows: the first part of transcription factor FoxO3a in hyperbaric oxygen induced acute oxygen poisoning in mice. For 6 ATA, 30 min hyperbaric oxygen exposure, established acute oxygen poisoning model. Through the observation of FoxO3a gene knockout mice and the latency of seizure frequency evaluation of brain injury and the pathological section and bronchoalveolar lavage fluid protein quantitative detection and evaluation of the damage of lung tissue, through the detection of anti oxidase activity, oxidation products to explore the role of FoxO3a in the index of the mechanism. The mice exposed to hyperbaric oxygen, the expression of FoxO3a Western blot was used to detect the cerebral cortex and lung tissue, immunohistochemical localization of FoxO3a and FoxO3a may explore on oxidative stress in the process of signal pathway and mechanism. Given normal mice PI3K/AKT inhibitor LY294002 blocking PI3K/Akt/FoxO3a pathway in brain tissue of mice the lung tissue and evaluation, in mice exposed to hyperbaric oxygen of brain tissue and lung tissue injury. The results showed that FoxO3a gene knockout mice convulsion latent Than not knockout mice significantly reduced, and the number of seizures within 30 min increased significantly, FoxO3a knockout mice lung injury is not generally serious knockout mice exposed to hyperbaric oxygen; after 8 h, FoxO3a protein content reached the peak, and the protein obviously into the nucleus; main signal pathway of PI3K/AKT inhibitors LY294002 inhibition of FoxO3a after FoxO3a was dephosphorylated, from cytoplasmic translocation into the nucleus, brain injury and lung injury relieved obviously. The second part of the transcription factor FoxO3a induced by chronic oxygen poisoning in the role of hyperbaric oxygen research mice were 2.5 ATA, 6 h hyperbaric oxygen exposure. The establishment of chronic oxygen poisoning model. The expression of FoxO3a protein and cytoplasm of detecting brain and lung tissues, the localization of the nucleus; PI3K/AKT inhibitor LY294002 PI3K/Akt/FoxO3a pathway in mice after hyperbaric oxygen exposure assessment The lung tissue injury. The results showed that HBO exposure after 8 h, FoxO3a protein content reached the peak, and changed into the nucleus process; PI3K/AKT inhibitor LY294002 induces FoxO3a phosphorylation, the cytoplasmic translocation into the nucleus, and alleviate lung injury. The results show that: FoxO3a can obviously reduce the hyperbaric oxygen induced acute and chronic oxygen poisoning; protein level can up regulate FoxO3a and promote the initiative to fight against nuclear oxygen poisoning hyperbaric oxygen exposure; inhibition of PI3K/AKT signaling makes FoxO3a into the nucleus, increased the activity of FoxO3a, brain and lung injury was obviously relieved, further confirmed FoxO3a is to enter the nucleus after play a protective role.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R595

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