Tat-NGB低氧神经保护作用机理研究及低氧相关模型探索与应用

发布时间：2018-06-11 22:39

本文选题：脑红蛋白 + 低氧　；参考：《中国人民解放军军事医学科学院》2016年博士论文

【摘要】：人体正常组织氧分压为2-9%(14-65 mm Hg),而吸入空气的氧分压为21%(160mm Hg),当外界空气中氧分压降低或机体氧气运输利用受阻引起血液及组织中氧分压低于正常值,不能满足组织器官的氧气需求,则引起组织缺氧。低氧是人及动物等生命体十分常见的一种应激条件,已有大量研究表明低氧可引起心脑血管系统、免疫系统、呼吸系统、消化系统和运动系统等诸多生命机能紊乱,例如高海拔地区低气压及低氧分压引起的急慢性高原病、新生儿缺氧诱发的癫痫、感染引起的低氧血症、局部供血障碍导致的脑梗等,这些常见的疾病均与组织或细胞缺氧密切相关。因此,关于低氧相关疾病的发病机理及干预方法一直是低氧领域研究的热点,对于预防和治疗低氧诱发疾病具有重要意义。本文围绕低氧这一主题展开,探讨了具有潜在低氧保护功能且融合穿膜肽HIV-1 Tat的Tat-NGB酵母体系表达及蛋白低氧保护功能、基于亚硫酸钠微生物低氧模型的建立及低氧保护作用机理以及在低氧环境中极易受到损伤的脑组织在极端营养条件下的自我调节机制。目前,对于低氧损伤保护剂的研究主要集中于具有自由基清除作用的多糖类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质具有一定的细胞损伤修复和保护作用,但由于如脑卒中等疾病发病迅速且有血脑屏障的阻碍,应用于临床的低氧损伤保护类制剂或药物极少。脑红蛋白(Neuroglobin,NGB)自2000年被发现以来受到极高关注,是主要分布在神经系统细胞和视网膜的第三类珠蛋白,与血红蛋白、肌红蛋白、胞红蛋白类似,具有携氧功能。大量关于NGB功能的研究显示低氧条件下神经元中的NGB表达量显著升高,同时NGB具有清除超氧自由基的能力,因此NGB具有潜在的抵御细胞低氧损伤的功能。而通过外源重组NGB提高神经元中NGB含量或可预防和治疗低氧对神经组织的损伤以及脑卒中等低氧相关疾病。因此,本文以NGB作为预防治疗神经细胞缺氧症的潜在药物蛋白,并尝试利用酵母表达系统实现重组蛋白高保真表达(除本研究外目前尚未见报道),同时融合表达HIV-1 Tat穿膜肽标签赋予重组蛋白穿膜功能以期克服血脑屏障的阻碍,并对重组蛋白的神经保护功能进行了验证。在低氧研究过程中,模拟低氧是一个重要的环节,目前常用的低氧培养箱等物理方法具有设备依赖性强、费用昂贵、操作复杂等缺点,而氯化钴、连二亚硫酸钠等化学低氧诱导剂又存在重金属毒性、不稳定遇水放热等不足之处,因此本文通过调研及前期工作参考将亚硫酸钠作为一种低氧诱导剂应用于微生物的低氧培养并初步探讨了大肠杆菌低氧调节的相关机制。大脑在机体缺氧时极易受到损伤,它在人及动物体中都是神经中枢,占有重要的地位,在低氧研究中也是重要的靶器官,对其应激机制研究也至关重要。基于对脑在应激条件下的应答策略和调节机制的关注,本文同时对此进行了相关研究。脑组织重量仅为机体比重的2%却要消耗20%的能量。传统理论认为大脑在能量利用方面具有自私性,自身储能极少,所需能量都是从周围神经组织获取甚至以损伤其他组织器官为代价。脑红蛋白的发现进一步佐证了这一观点,因其具有高效的携氧能力可保证脑组织的供能供养。而本文中从脑组织营养极端缺乏条件下水分调节及自噬角度进一步对脑在机体中的特殊地位进行了探讨。本文的主要研究成果及结论如下:1.重组脑红蛋白真核表达纯化及其对低氧损伤保护效应研究利用此前构建的人源脑红蛋白原核表达载体p ET28b-Ngb作为模板通过PCR扩增获取目的基因片段。Ngb目的基因与p PIC8k载体经Eco RI、Not I双酶切并连接后完成含Ngb基因的p PIC8k-Ngb载体构建。分别用SalⅠ、BglⅡ两种酶将载体线性化后电转化到宿主毕赤酵母菌GS115细胞后涂板利用MD培养板筛选阳性克隆,经SalⅠ、BglⅡ两种酶线性化的质粒均实现转化长出阳性克隆。随后经MM、MD培养板筛选出甲醇营养型的菌株,继而通过遗传霉素G418和菌落PCR筛选具有外源质粒高拷贝数的菌株、诱导表达并SDS-PAGE检测从中选出可高表达重组NGB蛋白的菌株用于后续蛋白纯化及功能研究。上述Ngb基因真核表达载体的构建及高产目的菌株的筛选过程,成功地实现了重组NGB蛋白在酵母表达体系中的表达。外源重组蛋白经真核细胞加工修饰更加保真了其原有功能,另外重组NGB蛋白可直接分泌到毕赤酵母培养液中简化了收集蛋白的复杂性,也避免了包涵形式无效蛋白的表达,为后续重组NGB的功能研究及应用研究奠定了基础。利用上述筛选出的高效表达NGB重组蛋白的酵母菌株采用Ni-NTA亲和层析柱将酵母表达上清液中的重组NGB蛋白富集纯化。纯化后的含Tat标签的Tat-NGB及不含Tat标签的NGB蛋白分别与PC12细胞共培养12 h后通过Western blot检测细胞内的NGB蛋白含量,结果发现His-NGB-His共培养细胞中未见重组NGB蛋白,而Tat-NGB-His组细胞中可检测到明显的重组NGB条带,说明HIV-1 Tat跨膜转导肽标签的融合表达使重组NGB蛋白具有穿膜功能。细胞中重组NGB蛋白在1.5 h内即可累积到稳定水平,进入细胞的速度可以满足低氧细胞干预治疗的时间窗。利用CCK-8试剂盒检测低氧环境培养细胞分别加入终浓度为5 m M Tat-NGB、NGB后细胞活性,结果显示低氧明显抑制了对照组和NGB组的细胞活性,而Tat-NGB组表现出显著提高的细胞活力。说明重组Tat-NGB不仅能穿膜进入神经细胞并且可以在细胞内发挥低氧损伤修复保护等作用。综上所述,本部分研究首次实现了重组NGB蛋白在毕赤酵母真核表达体系中的表达纯化。得到的重组NGB蛋白不仅保真了NGB蛋白原有的功能可有效的保护PC12细胞在低氧条件下的生长,而且还经由HIV-1 Tat标签实现了跨膜转导功能,为重组NGB作为神经保护药物的潜在应用奠定了前期实践基础。2.亚硫酸钠在微生物低氧诱导中的作用及相关机制研究首先利用微生物生长曲线仪检测不同浓度亚硫酸钠对不同大肠杆菌株系(BL21(DE3)、HB101、DH5α、OP50)发现,亚硫酸钠对大肠杆菌具有明显且广泛的生长抑制作用。继而测定E.coli生长过程中不同时间点的溶解氧含量确认亚硫酸钠对E.coli的生长抑制是由溶氧含量引起的,当Na2SO3浓度大于40 m M时培养体系的无氧状态可稳定维持长达8 h。通过透射电镜(TEM)观察Na2SO3抑制生长的E.coli菌体细胞形态,并未见明显的细胞破裂或损伤。综上所述Na2SO3可耗尽液体培养体系中的氧气快速达到与物理低氧相似的效果、无氧效果稳定且时间足够长、对菌体细胞无明显损伤、使用范围广,由此说明Na2SO3可以作为一种理想的低氧诱导剂应用于微生物厌氧培养及低氧研究。基于Na2SO3低氧模型,通过对低氧相关基因缺失的菌株检测发现,低氧相关通路基因在低氧应答及调节中所起的作用不同,其中fnr、arc B两个基因起到主要作用。考虑到动物肠道中细菌99%为厌氧菌,对Na2SO3的低氧效应研究进一步拓展到动物体内,通过灌胃的方式使Na2SO3溶液进入小鼠胃肠道,从而对亚硫酸钠在动物体内的作用效应进行了探讨,主要从机体整体氧化应激水平角度进行研究。研究结果显示,体内亚硫酸钠并未对动物的生长发育产生明显影响,但其引起小鼠体内脂质过氧化物MDA含量显著升高,同时SOD酶活性也相应增加。说明Na2SO3虽然其对小鼠整体的生长发育没有明显影响,但可通过氧化应激反应对小鼠细胞造成慢性损伤,对动物而言较高浓度Na2SO3溶液不适合用于其体内研究。然而对于动物肠道内的丰富菌群来说,其中99%为厌氧菌,依赖于肠道的低氧或无氧环境生存,Na2SO3消耗掉肠残余氧气或许有利于肠道菌群的生长及其有益生理作用的发挥,这一研究思路将成为后续研究的重点。在调研可用于低氧诱导的化学试剂时,考虑到最常用的Co Cl2具有严重的重金属毒性,后续的研究比较了钴等重金属离子对E.coli生长繁殖的抑制作用。研究发现重金属离子对E.coli的生长具有明显的损伤效应,但不同重金属离子的作用方式存在差异。3.小鼠脑组织在极度营养缺乏条件下自我调节及机理研究将小鼠分为禁食组、禁水组和水食双禁组,各组小鼠体重和血糖均随时间急剧下降,然而脑的重量却能相对保持稳定,且对脑组织进行HE染色观察显示那组织细胞形态正常并未出现明显的损伤,因此在禁食禁水这种极端营养条件下,脑组织可以实现自我保护维持其生理形态和功能。进一步通过RT-PCR的方法分析脑组织的水通道蛋白AQP-1、AQP-4的表达可见两基因的表达可基本维持正常水平以保证脑组织的正常而稳定的水含量。用Western Blot方法检测LC3蛋白表达以确定各组脑组织自噬水平高低,结果显示各实验组脑组织的自噬水平均有不同程度的提高,而水食双禁组表现的最为明显。综上本章内容研究了小鼠在饮食饮水限制的极端营养条件下脑组织自我保全的现象。并从含水量维持及平衡相关蛋白AQPs表达量角度解析了脑组织自我保全过程中水分平衡的机理。自噬蛋白的分析解释了脑组织在极端恶劣情况下除了与周边器官竞争能量的自私行为之外,也有通过自噬基于自身蛋白分解的自我拯救行为。亦即从含水量角度拓展了自私大脑的理论,并从自噬的非自私角度对自私大脑理论进行了补充。另外,在后续的研究中,AQP蛋白家族在营养不良等环境条件下对组织水含量的调控值得深入挖掘,可能为营养不良及相关症状的预防和治疗提供潜在的干预靶位和药物靶点,为低氧脑损伤研究也提供了相关的切入点。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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