本文選題：腦紅蛋白 + 低氧��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2016年博士論文

【摘要】：人體正常組織氧分壓為2-9%(14-65 mm Hg),而吸入空氣的氧分壓為21%(160mm Hg),當(dāng)外界空氣中氧分壓降低或機(jī)體氧氣運(yùn)輸利用受阻引起血液及組織中氧分壓低于正常值,不能滿足組織器官的氧氣需求,則引起組織缺氧。低氧是人及動(dòng)物等生命體十分常見(jiàn)的一種應(yīng)激條件,已有大量研究表明低氧可引起心腦血管系統(tǒng)、免疫系統(tǒng)、呼吸系統(tǒng)、消化系統(tǒng)和運(yùn)動(dòng)系統(tǒng)等諸多生命機(jī)能紊亂,例如高海拔地區(qū)低氣壓及低氧分壓引起的急慢性高原病、新生兒缺氧誘發(fā)的癲癇、感染引起的低氧血癥、局部供血障礙導(dǎo)致的腦梗等,這些常見(jiàn)的疾病均與組織或細(xì)胞缺氧密切相關(guān)。因此,關(guān)于低氧相關(guān)疾病的發(fā)病機(jī)理及干預(yù)方法一直是低氧領(lǐng)域研究的熱點(diǎn),對(duì)于預(yù)防和治療低氧誘發(fā)疾病具有重要意義。本文圍繞低氧這一主題展開(kāi),探討了具有潛在低氧保護(hù)功能且融合穿膜肽HIV-1 Tat的Tat-NGB酵母體系表達(dá)及蛋白低氧保護(hù)功能、基于亞硫酸鈉微生物低氧模型的建立及低氧保護(hù)作用機(jī)理以及在低氧環(huán)境中極易受到損傷的腦組織在極端營(yíng)養(yǎng)條件下的自我調(diào)節(jié)機(jī)制。目前,對(duì)于低氧損傷保護(hù)劑的研究主要集中于具有自由基清除作用的多糖類抗氧化物以及天然提取物等,如褐藻多糖等具有抗氧化性的物質(zhì)具有一定的細(xì)胞損傷修復(fù)和保護(hù)作用,但由于如腦卒中等疾病發(fā)病迅速且有血腦屏障的阻礙,應(yīng)用于臨床的低氧損傷保護(hù)類制劑或藥物極少。腦紅蛋白(Neuroglobin,NGB)自2000年被發(fā)現(xiàn)以來(lái)受到極高關(guān)注,是主要分布在神經(jīng)系統(tǒng)細(xì)胞和視網(wǎng)膜的第三類珠蛋白,與血紅蛋白、肌紅蛋白、胞紅蛋白類似,具有攜氧功能。大量關(guān)于NGB功能的研究顯示低氧條件下神經(jīng)元中的NGB表達(dá)量顯著升高,同時(shí)NGB具有清除超氧自由基的能力,因此NGB具有潛在的抵御細(xì)胞低氧損傷的功能。而通過(guò)外源重組NGB提高神經(jīng)元中NGB含量或可預(yù)防和治療低氧對(duì)神經(jīng)組織的損傷以及腦卒中等低氧相關(guān)疾病。因此,本文以NGB作為預(yù)防治療神經(jīng)細(xì)胞缺氧癥的潛在藥物蛋白,并嘗試?yán)媒湍副磉_(dá)系統(tǒng)實(shí)現(xiàn)重組蛋白高保真表達(dá)(除本研究外目前尚未見(jiàn)報(bào)道),同時(shí)融合表達(dá)HIV-1 Tat穿膜肽標(biāo)簽賦予重組蛋白穿膜功能以期克服血腦屏障的阻礙,并對(duì)重組蛋白的神經(jīng)保護(hù)功能進(jìn)行了驗(yàn)證。在低氧研究過(guò)程中,模擬低氧是一個(gè)重要的環(huán)節(jié),目前常用的低氧培養(yǎng)箱等物理方法具有設(shè)備依賴性強(qiáng)、費(fèi)用昂貴、操作復(fù)雜等缺點(diǎn),而氯化鈷、連二亞硫酸鈉等化學(xué)低氧誘導(dǎo)劑又存在重金屬毒性、不穩(wěn)定遇水放熱等不足之處,因此本文通過(guò)調(diào)研及前期工作參考將亞硫酸鈉作為一種低氧誘導(dǎo)劑應(yīng)用于微生物的低氧培養(yǎng)并初步探討了大腸桿菌低氧調(diào)節(jié)的相關(guān)機(jī)制。大腦在機(jī)體缺氧時(shí)極易受到損傷,它在人及動(dòng)物體中都是神經(jīng)中樞,占有重要的地位,在低氧研究中也是重要的靶器官,對(duì)其應(yīng)激機(jī)制研究也至關(guān)重要�；趯�(duì)腦在應(yīng)激條件下的應(yīng)答策略和調(diào)節(jié)機(jī)制的關(guān)注,本文同時(shí)對(duì)此進(jìn)行了相關(guān)研究。腦組織重量?jī)H為機(jī)體比重的2%卻要消耗20%的能量。傳統(tǒng)理論認(rèn)為大腦在能量利用方面具有自私性,自身儲(chǔ)能極少,所需能量都是從周?chē)窠?jīng)組織獲取甚至以損傷其他組織器官為代價(jià)。腦紅蛋白的發(fā)現(xiàn)進(jìn)一步佐證了這一觀點(diǎn),因其具有高效的攜氧能力可保證腦組織的供能供養(yǎng)。而本文中從腦組織營(yíng)養(yǎng)極端缺乏條件下水分調(diào)節(jié)及自噬角度進(jìn)一步對(duì)腦在機(jī)體中的特殊地位進(jìn)行了探討。本文的主要研究成果及結(jié)論如下:1.重組腦紅蛋白真核表達(dá)純化及其對(duì)低氧損傷保護(hù)效應(yīng)研究利用此前構(gòu)建的人源腦紅蛋白原核表達(dá)載體p ET28b-Ngb作為模板通過(guò)PCR擴(kuò)增獲取目的基因片段。Ngb目的基因與p PIC8k載體經(jīng)Eco RI、Not I雙酶切并連接后完成含Ngb基因的p PIC8k-Ngb載體構(gòu)建。分別用SalⅠ、BglⅡ兩種酶將載體線性化后電轉(zhuǎn)化到宿主畢赤酵母菌GS115細(xì)胞后涂板利用MD培養(yǎng)板篩選陽(yáng)性克隆,經(jīng)SalⅠ、BglⅡ兩種酶線性化的質(zhì)粒均實(shí)現(xiàn)轉(zhuǎn)化長(zhǎng)出陽(yáng)性克隆。隨后經(jīng)MM、MD培養(yǎng)板篩選出甲醇營(yíng)養(yǎng)型的菌株,繼而通過(guò)遺傳霉素G418和菌落PCR篩選具有外源質(zhì)粒高拷貝數(shù)的菌株、誘導(dǎo)表達(dá)并SDS-PAGE檢測(cè)從中選出可高表達(dá)重組NGB蛋白的菌株用于后續(xù)蛋白純化及功能研究。上述Ngb基因真核表達(dá)載體的構(gòu)建及高產(chǎn)目的菌株的篩選過(guò)程,成功地實(shí)現(xiàn)了重組NGB蛋白在酵母表達(dá)體系中的表達(dá)。外源重組蛋白經(jīng)真核細(xì)胞加工修飾更加保真了其原有功能,另外重組NGB蛋白可直接分泌到畢赤酵母培養(yǎng)液中簡(jiǎn)化了收集蛋白的復(fù)雜性,也避免了包涵形式無(wú)效蛋白的表達(dá),為后續(xù)重組NGB的功能研究及應(yīng)用研究奠定了基礎(chǔ)。利用上述篩選出的高效表達(dá)NGB重組蛋白的酵母菌株采用Ni-NTA親和層析柱將酵母表達(dá)上清液中的重組NGB蛋白富集純化。純化后的含Tat標(biāo)簽的Tat-NGB及不含Tat標(biāo)簽的NGB蛋白分別與PC12細(xì)胞共培養(yǎng)12 h后通過(guò)Western blot檢測(cè)細(xì)胞內(nèi)的NGB蛋白含量,結(jié)果發(fā)現(xiàn)His-NGB-His共培養(yǎng)細(xì)胞中未見(jiàn)重組NGB蛋白,而Tat-NGB-His組細(xì)胞中可檢測(cè)到明顯的重組NGB條帶,說(shuō)明HIV-1 Tat跨膜轉(zhuǎn)導(dǎo)肽標(biāo)簽的融合表達(dá)使重組NGB蛋白具有穿膜功能。細(xì)胞中重組NGB蛋白在1.5 h內(nèi)即可累積到穩(wěn)定水平,進(jìn)入細(xì)胞的速度可以滿足低氧細(xì)胞干預(yù)治療的時(shí)間窗。利用CCK-8試劑盒檢測(cè)低氧環(huán)境培養(yǎng)細(xì)胞分別加入終濃度為5 m M Tat-NGB、NGB后細(xì)胞活性,結(jié)果顯示低氧明顯抑制了對(duì)照組和NGB組的細(xì)胞活性,而Tat-NGB組表現(xiàn)出顯著提高的細(xì)胞活力。說(shuō)明重組Tat-NGB不僅能穿膜進(jìn)入神經(jīng)細(xì)胞并且可以在細(xì)胞內(nèi)發(fā)揮低氧損傷修復(fù)保護(hù)等作用。綜上所述,本部分研究首次實(shí)現(xiàn)了重組NGB蛋白在畢赤酵母真核表達(dá)體系中的表達(dá)純化。得到的重組NGB蛋白不僅保真了NGB蛋白原有的功能可有效的保護(hù)PC12細(xì)胞在低氧條件下的生長(zhǎng),而且還經(jīng)由HIV-1 Tat標(biāo)簽實(shí)現(xiàn)了跨膜轉(zhuǎn)導(dǎo)功能,為重組NGB作為神經(jīng)保護(hù)藥物的潛在應(yīng)用奠定了前期實(shí)踐基礎(chǔ)。2.亞硫酸鈉在微生物低氧誘導(dǎo)中的作用及相關(guān)機(jī)制研究首先利用微生物生長(zhǎng)曲線儀檢測(cè)不同濃度亞硫酸鈉對(duì)不同大腸桿菌株系(BL21(DE3)、HB101、DH5α、OP50)發(fā)現(xiàn),亞硫酸鈉對(duì)大腸桿菌具有明顯且廣泛的生長(zhǎng)抑制作用。繼而測(cè)定E.coli生長(zhǎng)過(guò)程中不同時(shí)間點(diǎn)的溶解氧含量確認(rèn)亞硫酸鈉對(duì)E.coli的生長(zhǎng)抑制是由溶氧含量引起的,當(dāng)Na2SO3濃度大于40 m M時(shí)培養(yǎng)體系的無(wú)氧狀態(tài)可穩(wěn)定維持長(zhǎng)達(dá)8 h。通過(guò)透射電鏡(TEM)觀察Na2SO3抑制生長(zhǎng)的E.coli菌體細(xì)胞形態(tài),并未見(jiàn)明顯的細(xì)胞破裂或損傷。綜上所述Na2SO3可耗盡液體培養(yǎng)體系中的氧氣快速達(dá)到與物理低氧相似的效果、無(wú)氧效果穩(wěn)定且時(shí)間足夠長(zhǎng)、對(duì)菌體細(xì)胞無(wú)明顯損傷、使用范圍廣,由此說(shuō)明Na2SO3可以作為一種理想的低氧誘導(dǎo)劑應(yīng)用于微生物厭氧培養(yǎng)及低氧研究�；贜a2SO3低氧模型,通過(guò)對(duì)低氧相關(guān)基因缺失的菌株檢測(cè)發(fā)現(xiàn),低氧相關(guān)通路基因在低氧應(yīng)答及調(diào)節(jié)中所起的作用不同,其中fnr、arc B兩個(gè)基因起到主要作用�？紤]到動(dòng)物腸道中細(xì)菌99%為厭氧菌,對(duì)Na2SO3的低氧效應(yīng)研究進(jìn)一步拓展到動(dòng)物體內(nèi),通過(guò)灌胃的方式使Na2SO3溶液進(jìn)入小鼠胃腸道,從而對(duì)亞硫酸鈉在動(dòng)物體內(nèi)的作用效應(yīng)進(jìn)行了探討,主要從機(jī)體整體氧化應(yīng)激水平角度進(jìn)行研究。研究結(jié)果顯示,體內(nèi)亞硫酸鈉并未對(duì)動(dòng)物的生長(zhǎng)發(fā)育產(chǎn)生明顯影響,但其引起小鼠體內(nèi)脂質(zhì)過(guò)氧化物MDA含量顯著升高,同時(shí)SOD酶活性也相應(yīng)增加。說(shuō)明Na2SO3雖然其對(duì)小鼠整體的生長(zhǎng)發(fā)育沒(méi)有明顯影響,但可通過(guò)氧化應(yīng)激反應(yīng)對(duì)小鼠細(xì)胞造成慢性損傷,對(duì)動(dòng)物而言較高濃度Na2SO3溶液不適合用于其體內(nèi)研究。然而對(duì)于動(dòng)物腸道內(nèi)的豐富菌群來(lái)說(shuō),其中99%為厭氧菌,依賴于腸道的低氧或無(wú)氧環(huán)境生存,Na2SO3消耗掉腸殘余氧氣或許有利于腸道菌群的生長(zhǎng)及其有益生理作用的發(fā)揮,這一研究思路將成為后續(xù)研究的重點(diǎn)。在調(diào)研可用于低氧誘導(dǎo)的化學(xué)試劑時(shí),考慮到最常用的Co Cl2具有嚴(yán)重的重金屬毒性,后續(xù)的研究比較了鈷等重金屬離子對(duì)E.coli生長(zhǎng)繁殖的抑制作用。研究發(fā)現(xiàn)重金屬離子對(duì)E.coli的生長(zhǎng)具有明顯的損傷效應(yīng),但不同重金屬離子的作用方式存在差異。3.小鼠腦組織在極度營(yíng)養(yǎng)缺乏條件下自我調(diào)節(jié)及機(jī)理研究將小鼠分為禁食組、禁水組和水食雙禁組,各組小鼠體重和血糖均隨時(shí)間急劇下降,然而腦的重量卻能相對(duì)保持穩(wěn)定,且對(duì)腦組織進(jìn)行HE染色觀察顯示那組織細(xì)胞形態(tài)正常并未出現(xiàn)明顯的損傷,因此在禁食禁水這種極端營(yíng)養(yǎng)條件下,腦組織可以實(shí)現(xiàn)自我保護(hù)維持其生理形態(tài)和功能。進(jìn)一步通過(guò)RT-PCR的方法分析腦組織的水通道蛋白AQP-1、AQP-4的表達(dá)可見(jiàn)兩基因的表達(dá)可基本維持正常水平以保證腦組織的正常而穩(wěn)定的水含量。用Western Blot方法檢測(cè)LC3蛋白表達(dá)以確定各組腦組織自噬水平高低,結(jié)果顯示各實(shí)驗(yàn)組腦組織的自噬水平均有不同程度的提高,而水食雙禁組表現(xiàn)的最為明顯。綜上本章內(nèi)容研究了小鼠在飲食飲水限制的極端營(yíng)養(yǎng)條件下腦組織自我保全的現(xiàn)象。并從含水量維持及平衡相關(guān)蛋白AQPs表達(dá)量角度解析了腦組織自我保全過(guò)程中水分平衡的機(jī)理。自噬蛋白的分析解釋了腦組織在極端惡劣情況下除了與周邊器官競(jìng)爭(zhēng)能量的自私行為之外,也有通過(guò)自噬基于自身蛋白分解的自我拯救行為。亦即從含水量角度拓展了自私大腦的理論,并從自噬的非自私角度對(duì)自私大腦理論進(jìn)行了補(bǔ)充。另外,在后續(xù)的研究中,AQP蛋白家族在營(yíng)養(yǎng)不良等環(huán)境條件下對(duì)組織水含量的調(diào)控值得深入挖掘,可能為營(yíng)養(yǎng)不良及相關(guān)癥狀的預(yù)防和治療提供潛在的干預(yù)靶位和藥物靶點(diǎn),為低氧腦損傷研究也提供了相關(guān)的切入點(diǎn)。
[Abstract]:The oxygen partial pressure in normal tissues of the human body is 2-9% (14-65 mm Hg), while the oxygen partial pressure of the inhaled air is 21% (160mm Hg). When the oxygen partial pressure in the air is reduced or the oxygen transport and utilization of the body is blocked, the oxygen partial pressure in the blood and tissue is lower than the normal value, and the oxygen demand of the tissues and organs can not be met. The hypoxia is the life of human and animal. A very common stress condition, a large number of studies have shown that hypoxia can cause the cardiovascular system, the immune system, the respiratory system, the digestive system, and the motor system and many other life disorders, such as the acute and chronic plateau disease caused by low pressure and hypoxic pressure at high altitude, the epilepsy induced by hypoxia in the newborn, the low infection caused by infection. These common diseases are closely related to tissue or cell hypoxia. Therefore, the pathogenesis and intervention methods of hypoxic related diseases have always been the hot spots in the field of hypoxia. It is of great significance for the prevention and treatment of hypoxia induced disease. This article focuses on the theme of hypoxia. The expression of Tat-NGB yeast system with membrane peptide HIV-1 Tat and protective function of protein hypoxia, based on the establishment of sodium sulfite microbiological hypoxia model and the mechanism of hypoxic protection, and the self regulating machine of brain tissue which are easily damaged in the low oxygen environment under extreme nutritional conditions. At present, the study of hypoxic damage protectants is mainly focused on the polysaccharide antioxidants and natural extracts with free radical scavenging effects, such as brown algae polysaccharide, and other substances with antioxidant properties, which have a certain cell damage repair and protection, but are rapid and have a blood brain barrier due to diseases such as stroke. Neuroglobin (NGB) has received high attention since its discovery in 2000. It is mainly distributed in the third kinds of globin in the cells of the nervous system and the retina, similar to hemoglobin, myoglobin, and cytosolic protein, and has a large amount of function of NGB. The study showed that the expression of NGB in neurons was significantly higher in hypoxic neurons, while NGB had the ability to scavenge superoxide radicals. Therefore, NGB had the potential to resist the damage of hypoxia in cells. By exogenous recombinant NGB, the content of NGB in neurons could be improved or the injury of hypoxia to the nerve tissue and the moderate hypoxia of cerebral apoplexy could be prevented and treated. Therefore, NGB is used as a potential drug protein for the prevention and treatment of neuro anoxia, and the yeast expression system is used to realize the high fidelity expression of recombinant protein (but it has not yet been reported in this study). At the same time, the fusion expression of HIV-1 Tat membrane peptide labeling is given to the recombinant protein transmembrane function in order to overcome the blood brain barrier. The neuroprotective function of the recombinant protein is verified. In the study of hypoxia, simulated hypoxia is an important link. The current physical methods, such as low oxygen incubator, have the disadvantages of strong equipment dependence, expensive cost and complicated operation, while cobalt chloride and two sodium sulfite and other chemical hypoxia inducers have heavy gold. In this paper, we apply sodium sulfite as a hypoxic inducer to the hypoxic culture of microorganism and preliminarily discuss the mechanism of hypoxia regulation in Escherichia coli. The brain is easily damaged when the body is anoxic, it is both in human and animal body. The nerve center, which occupies an important position, is also an important target organ in the study of hypoxia. It is also important to study the mechanism of stress. Based on the attention of the brain in the response and regulation mechanism under stress conditions, this paper has carried out a related study at the same time. The weight of brain tissue is only 2% of the body's specific gravity but consumes 20% of the energy. The theory is that the brain is selfish in energy use, and its energy is very small. The energy required is at the expense of the peripheral nerve tissue and even the damage to other tissues and organs. The discovery of the brain erythrocyte is further supported by the discovery of the high energy carrying capacity of the brain which can guarantee the supply of brain tissue. The special status of brain in the body was further studied by water regulation and autophagy. The main research results and conclusions of this paper are as follows: 1. recombinant eukaryotic expression and purification of recombinant brain erythrocyte and its protective effect on hypoxia injury by using the previously constructed human brain erythroprotein prokaryotic expression vector p ET 28b-Ngb as a template, the target gene fragment.Ngb was amplified by PCR and P PIC8k vector was constructed by Eco RI, Not I double enzyme cut and connected to complete the P PIC8k-Ngb carrier containing Ngb gene. The positive clones were transformed into positive clones by Sal I, Bgl II two enzyme linearized plasmids. Then the strains of methanol nutrition were screened by MM and MD culture plate, then the strains with high copies of exogenous plasmid were screened by genetic mycophenycin G418 and colony PCR, and the high expression of recombinant NGB eggs was selected and expressed by SDS-PAGE detection. The white strain is used for subsequent protein purification and functional study. The construction of the eukaryotic expression vector of the Ngb gene and the screening process of high yield strains have successfully realized the expression of the recombinant NGB protein in the yeast expression system. The recombinant protein has been reconstituted by eukaryotic cell processing and reconstituted the original function of the recombinant protein, and the recombinant NGB protein can be reorganized. Directly secreted into Pichia pastoris culture solution simplifies the complexity of protein collection, and avoids the expression of inclusion form invalid protein. It lays the foundation for the functional study and application of the subsequent recombinant NGB. The yeast strain with high efficiency expressing the recombinant protein of NGB is used to express the yeast by Ni-NTA affinity chromatography column. The recombinant NGB protein in the liquid was enriched and purified. The purified Tat labeled Tat-NGB and the NGB protein without the Tat label were co cultured with PC12 cells for 12 h respectively, and the NGB protein content in the cells was detected by Western blot. The results showed that the recombinant NGB protein was not found in the His-NGB-His co culture cells, and the cells in the group could be detected to be obvious. The recombinant NGB band shows that the fusion expression of the HIV-1 Tat transmembrane transduction peptide label makes the recombinant NGB protein have the membrane function. The recombinant NGB protein in the cell can accumulate to the stable level within 1.5 h, and the speed of entering the cell can meet the time window of the hypoxia cell intervention treatment. The cultured cells of the hypoxia environment are added to the culture cells of the hypoxia to be added to the cells respectively. The final concentration was 5 m M Tat-NGB, NGB after the cell activity. The results showed that hypoxia significantly inhibited the cell activity of the control group and NGB group, and the Tat-NGB group showed significant increase in cell viability. It indicated that the recombinant Tat-NGB could not only penetrate the membrane into the nerve cells but also play a role in the repair and protection of hypoxic damage in the cells. The expression and purification of recombinant NGB protein in the eukaryotic expression system of Pichia pastoris was realized for the first time. The recombinant NGB protein not only protects the original function of NGB protein to protect the growth of PC12 cells under the condition of hypoxia, but also realizes the transmembrane transduction function via the HIV-1 Tat label, and the recombinant NGB is used as the nerve protection. The potential application of drugs has laid the basis for the early practice of.2. sodium sulfite in the induction of microbial hypoxia induction and related mechanisms. First, microbial growth Qu Xianyi was used to detect different concentrations of sodium sulfite to different Escherichia coli strains (BL21 (DE3), HB101, DH5 a, OP50). Growth inhibition. Then determine the dissolved oxygen content at different time points in E.coli growth. It is confirmed that the growth inhibition of sodium sulfite on E.coli is caused by the dissolved oxygen content. When the Na2SO3 concentration is greater than 40 m M, the oxygen free state of the culture system can be maintained for up to 8 h. through the transmission electrical mirror (TEM) to observe the E.coli bacteria that inhibit the growth of Na2SO3 To sum up, the oxygen in the Na2SO3 depleted liquid culture system is similar to the physical hypoxia. The oxygen free effect is stable and the time is long enough. There is no obvious damage to the cell and the wide range of use. Thus, Na2SO3 can be used as an ideal hypoxia inducer. Applied to microbiological anaerobic culture and hypoxia study. Based on the Na2SO3 hypoxia model, it was found that the role of hypoxia related pathway gene in hypoxia response and regulation was different by detection of low oxygen related gene deletion, of which the two genes of FNR and arc B played a major role. Considering that the bacteria in the intestinal tract were 99% anaerobes and Na2SO 3 the study of hypoxic effect was further extended to the animal body. The effect of Na2SO3 solution into the gastrointestinal tract of mice was made by gavage, and the effect of sodium sulfite in the animal was discussed, mainly from the angle of the overall oxidative stress level of the body. The results showed that the body of sodium sulfite in the body did not grow on the animal. There was a significant effect on development, but the content of lipid peroxide MDA in mice increased significantly and the activity of SOD enzyme increased correspondingly. Although Na2SO3 had no obvious effect on the growth and development of mice, it could cause slow damage to mice cells by oxidative stress reaction, and the higher concentration of Na2SO3 solution for animals was not. It is suitable for the study in vivo. However, for the abundance of bacteria in the intestines of animals, 99% of them are anaerobic bacteria, depending on the low oxygen or anaerobic environment of the intestinal tract. Na2SO3 consumption of the residual oxygen in the intestines may be beneficial to the growth of intestinal microflora and its beneficial physiological functions. This study will be the focus of follow-up research. The investigation can be used for the chemical reagents induced by hypoxia, considering that the most commonly used Co Cl2 has serious heavy metal toxicity. Subsequent studies have compared the inhibitory effects of cobalt and other heavy metal ions on the growth and propagation of E.coli. The study found that heavy metal ions have obvious damage effects on the growth of E.coli, but the action modes of different heavy metal ions have been found. The self-regulation and mechanism study of brain tissue of.3. mice in the condition of extreme nutritional deficiency was divided into fasting group, water ban group and water feeding double ban group. The weight and blood sugar of mice in each group decreased sharply with time, but the weight of the brain remained relatively stable, and the HE staining of the brain group showed the morphology of the tissue cells. There is no obvious damage to normal, so the brain tissue can maintain its physiological form and function under the extreme nutrition condition of prohibition of water and water prohibition. The RT-PCR method is used to analyze the water channel protein AQP-1 of the brain tissue. The expression of AQP-4 can be seen that the expression of the two gene can maintain the normal level to guarantee the brain tissue. The Western Blot method was used to detect the expression of LC3 protein to determine the level of autophagy in each group of brain tissues. The results showed that the level of autophagy in the brain tissues of each group was improved in varying degrees, while the water food double forbidden group was the most obvious. The mechanism of water balance in the process of self preservation of brain tissue is analyzed from the maintenance of water content and the expression of balance related protein AQPs. The analysis of autophagic protein explains that the brain tissue is also through self - selfishness in extreme adverse circumstances except for the competitive energy with the peripheral organs. Self saving behavior based on self protein decomposition. That is, the theory of selfish brain is expanded from the water content angle, and the selfish brain theory is supplemented from the non selfishness angle of autophagy. In the follow-up study, the regulation of the water content of the AQP protein family under the condition of malnutrition is worth digging deeply. It can provide potential intervention targets and drug targets for the prevention and treatment of malnutrition and related symptoms, and also provides research for hypoxic brain injury.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R96

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