本文關(guān)鍵詞：轉(zhuǎn)錄調(diào)節(jié)因子FoxO3a在高壓氧致急、慢性氧中毒中的作用研究　出處：《第二軍醫(yī)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 高壓氧 急性氧中毒 慢性氧中毒 FoxO3a AKT PI3K

【摘要】：人們把超過一個大氣壓的壓力叫做高氣壓,將機體置于高氣壓環(huán)境中,呼吸純氧治療疾病的方法稱為高壓氧(HBO)療法。高壓氧療法已經(jīng)被廣泛地應(yīng)用于潛水作業(yè)、潛艇脫險和臨床相關(guān)疾病的治療。在潛水作業(yè)后,呼吸高壓氧進行減壓,可使?jié)撍^程中體內(nèi)產(chǎn)生的惰性氣體快速被置換出來,從而有效縮短減壓時間,提高工作效率,保護潛水員的生命安全。潛艇失事后,潛艇艇員在緊急情況下,預(yù)防性吸入高壓氧后再進行快速漂浮脫險,可以確保艇員生命安全,同時顯著減輕減壓病的病情及后遺癥。在臨床上,高壓氧能夠改善組織缺氧狀況,促進損傷組織的修復(fù)和再生,對高血壓腦出血、糖尿病足、脊髓損傷等具有一定的療效。但是機體持續(xù)吸入氧分壓過高的氣體后,可能會出現(xiàn)毒性反應(yīng)。當氧分壓200kPa時,毒性反應(yīng)會在較短時間內(nèi)發(fā)生,并以中樞神經(jīng)系統(tǒng)(CNS)功能紊亂為主要表現(xiàn),稱為中樞神經(jīng)系統(tǒng)氧中毒(CNS-OT)或急性氧中毒(acute oxygen toxicity),其最劇烈、最典型的表現(xiàn)是類似于癲癇大發(fā)作樣的全身強直性痙攣,即驚厥大發(fā)作,也稱為“氧驚厥”。氧驚厥發(fā)生的同時,機體也會出現(xiàn)以肺出血為主要表現(xiàn)的急性肺損傷。當氧分壓在60~200 kPa時,氧中毒損傷的主要靶器官是肺組織,主要表現(xiàn)為類似于支氣管肺炎的癥狀,稱為慢性氧中毒(chronic oxygen toxicity)。氧中毒的發(fā)生嚴重限制了高壓氧的應(yīng)用,深入研究氧中毒的發(fā)生發(fā)展過程及其發(fā)病機制,探索其致病的關(guān)鍵因素,對于有效預(yù)防氧中毒的發(fā)生具有非常重要的現(xiàn)實意義。近年的研究表明,轉(zhuǎn)錄調(diào)節(jié)因子FoxO3a在氧化應(yīng)激中具有非常重要的作用。FoxO3a既能誘導(dǎo)細胞在氧化應(yīng)激條件下凋亡,也能保護細胞在氧化應(yīng)激條件下存活,且不同的細胞、組織在不同的氧化應(yīng)激條件下,可能通過不同的機制發(fā)揮著多樣的功能。PI3K-Akt-FoxO3a信號通路在氧化應(yīng)激過程中發(fā)揮著重要的作用,被認為是FoxO3a調(diào)節(jié)氧化應(yīng)激的一個重要信號通路。在PI3K/Akt/FoxO3a信號通路中,FoxO3a的蘇氨酸殘基32(Thr32)、絲氨酸殘基253(Ser253)和絲氨酸殘基315(Ser315)位點發(fā)生磷酸化后,可以介導(dǎo)FoxO3a由胞核轉(zhuǎn)移到胞漿,轉(zhuǎn)錄活性受到抑制,下游相應(yīng)的生物學(xué)效應(yīng)也隨之改變。當PI3K/Akt/FoxO3a途徑受到抑制時,去磷酸化的FoxO3a大部分在細胞核聚集,激活下游靶基因的表達,進而調(diào)控相關(guān)的生物學(xué)功能。本課題旨在通過將小鼠經(jīng)過高壓氧暴露后觀察其腦組織和肺組織的病理變化及相關(guān)氧化指標的變化,探索FoxO3a在急、慢性氧中毒的發(fā)生發(fā)展過程中是否發(fā)揮作用。同時探討和確證FoxO3a在急、慢性氧中毒發(fā)生發(fā)展過程中的作用及其可能的機制,我們開展的研究和取得的結(jié)果主要有:第一部分轉(zhuǎn)錄調(diào)節(jié)因子FoxO3a在高壓氧致急性氧中毒中的作用研究將小鼠進行了6 ATA,30 min的高壓氧暴露,建立了急性氧中毒模型。通過觀察FoxO3a基因敲除小鼠驚厥潛伏期和發(fā)作次數(shù)評估腦損傷情況,利用病理學(xué)切片和肺泡灌洗液中蛋白定量檢測評估了肺組織的損傷情況,通過檢測抗氧化酶活性、氧化產(chǎn)物指標等探索FoxO3a在其中的作用機制。小鼠高壓氧暴露后,Western blot法檢測大腦皮層和肺組織中FoxO3a蛋白表達情況,免疫組化定位FoxO3a,探索FoxO3a在氧化應(yīng)激過程中可能的信號通路及機制。給予正常小鼠PI3K/AKT抑制劑LY294002阻斷小鼠腦組織和肺組織的PI3K/Akt/FoxO3a通路,評估小鼠在高壓氧暴露后腦組織和肺組織損傷情況。結(jié)果發(fā)現(xiàn),FoxO3a基因敲除小鼠的驚厥潛伏期比未敲除小鼠顯著地縮短,且30 min內(nèi)驚厥的次數(shù)明顯增多,FoxO3a基因敲除小鼠肺部總體損傷情況較未敲除小鼠嚴重;高壓氧暴露結(jié)束后8 h,FoxO3a蛋白含量達到峰值,且蛋白發(fā)生了明顯的入核;PI3K/AKT抑制劑LY294002抑制FoxO3a的主要信號通路后,FoxO3a明顯被去磷酸化,從胞漿轉(zhuǎn)位進入胞核中,腦損傷和肺組織損傷情況明顯緩解。第二部分轉(zhuǎn)錄調(diào)節(jié)因子FoxO3a在高壓氧致慢性氧中毒中的作用研究將小鼠進行了2.5 ATA,6 h的高壓氧暴露,建立了慢性氧中毒模型。檢測大腦皮層和肺組織中FoxO3a蛋白表達情況和胞漿、胞核的定位情況;PI3K/AKT抑制劑LY294002阻斷PI3K/Akt/FoxO3a通路,評估小鼠在高壓氧暴露后肺組織損傷情況。結(jié)果發(fā)現(xiàn),高壓氧暴露結(jié)束后8 h,FoxO3a蛋白含量達到峰值,且發(fā)生了明顯的入核過程;PI3K/AKT抑制劑LY294002使FoxO3a發(fā)生去磷酸化,使其從胞漿轉(zhuǎn)位進入胞核中,進而緩解肺組織損傷情況。研究結(jié)果表明:FoxO3a能夠明顯減輕高壓氧引起的急、慢性氧中毒;高壓氧暴露能夠主動上調(diào)FoxO3a的蛋白水平和促使其入核來對抗氧中毒的發(fā)生;抑制PI3K/AKT信號通路使得FoxO3a發(fā)生入核,提高了FoxO3a的活性,腦和肺損傷情況明顯緩解,進一步確證FoxO3a是進入胞核后發(fā)揮保護作用的。
[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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R595

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1 張亞楠;轉(zhuǎn)錄調(diào)節(jié)因子FoxO3a在高壓氧致急、慢性氧中毒中的作用研究[D];第二軍醫(yī)大學(xué);2016年

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本文編號：1389855