脑红蛋白抗自由基损伤机制的初步研究

发布时间：2018-04-07 15:13

本文选题：脑红蛋白　切入点：抗氧化　出处：《中国人民解放军军事医学科学院》2010年硕士论文

【摘要】： 自由基是机体正常代谢的中间产物,在生理状态下人体内自由基始终处于产生与清除的动态平衡过程中,而此平衡主要依靠机体内的抗氧化系统来维持。然而,过量的自由基是产生氧化应激的重要因素,在机体内较易与各种生物大分子发生反应而导致细胞和组织氧化损伤。机体内过多的自由基同时还是许多疾病产生的重要原因,如神经系统退行性疾病、脑中风、肿瘤等,因此清除过量自由基已成为防治此类疾病的重要策略。脑红蛋白(neuroglobin,Ngb)是Burmester于2000年新发现的第三类携氧珠蛋白,主要表达于神经系统,与氧有很高的亲和力,能促进氧传递到线粒体,提高脑组织对氧的利用率,可作为内源性神经保护因子保护神经元抵御缺血/缺氧性损伤。很多研究发现,具有携氧能力的珠蛋白Ngb与清除过量自由基有关,这说明Ngb在清除过多自由基方面可能起重要作用,但目前多数报道是通过真核细胞转染技术和转基因等手段,证明Ngb能够清除过量的有害自由基,保护细胞和组织抵抗自由基损伤。那么,Ngb蛋白是否直接具有清除自由基作用,其机理如何?这一问题尚未得到明确回答。本文拟对此进行重点研究。首先,利用体外和细胞内测定抗氧化剂清除自由基活性的研究方法,对重组人源脑红蛋白(recombinant human neuroglobin, rhNgb)的抗氧化能力、清除自由基活性、金属螯合能力以及对DNA氧化损伤的保护作用进行测定。考虑到Ngb作为携氧珠蛋白家族中的一员,其抗氧化能力是一个比较特定的功能,我们分别采用ABTS法和铁氰化钾还原法对rhNgb的抗氧化和还原能力进行测定,结果发现rhNgb具有一定的抗氧化能力,但其抗氧化能力要低于常规抗氧化剂Vc、GSH以及NAC;而还原能力测定结果表明,rhNgb几乎无还原能力,这可能与rhNgb本身的氧化还原状态有关。由于抗氧化剂清除氧自由基的活性是其抗氧化属性的重要指标,因此在研究rhNgb抗氧化和还原能力的同时,我们对其清除超氧阴离子、过氧化氢、羟自由基等进行了测定。本实验中rhNgb清除超氧阴离子、过氧化氢、羟自由基活性的检测分别采用四唑硝基蓝(nitro blue tetrazolium,NBT)-光还原法、H2O2灭活酶法、邻二氮菲-Fe(II)比色法进行。结果表明,rhNgb具有明确的清除超氧阴离子、过氧化氢、羟自由基等活性。进一步分析可知,rhNgb对超氧阴离子有很强的清除能力,其活性与Vc相差不大;rhNgb对过氧化氢的清除活性也较强,但其清除羟自由基能力较弱,与Vc和NAC相比有一定差距。另外,我们还对rhNgb的过氧化物酶活性和清除DPPH自由基能力进行了检测。结果发现,rhNgb几乎没有过氧化物酶活性,也没有清除DPPH自由基的能力。在证明rhNgb具有上述清除自由基活性的同时,为了更好地研究Ngb在抗自由基损伤方面的其他活性,我们还对rhNgb的Fe(II)螯合能力和对DNA氧化损伤的保护作用进行了探讨。过渡金属在机体内易于发生Fenton反应,产生羟自由基,因此抗氧化剂的金属螯合能力也是其抗氧化能力的一种体现。结果发现,rhNgb对过渡金属Fe(II)具有一定的螯合能力,但其螯合能力并非完全的浓度依赖性,在低浓度rhNgb时,其金属铁离子的螯合能力随着rhNgb浓度升高而增加,当rhNgb达到一定浓度时,其螯合能力开始下降。这可能与rhNgb中存在的铁卟啉环有一定的关系。为了更好地理解rhNgb是否对细胞内的自由基也具有清除活性,我们对rhNgb的细胞毒性和清除细胞过量活性氧(reactive oxygen species,ROS)进行了检测。目前对细胞内ROS检测方法有很多,从灵敏度等角度出发,我们选用灵敏度较高的DCFH-DA荧光探针法对rhNgb清除PC12细胞内过量ROS能力进行了研究。同时,为了便于确定rhNgb对PC12细胞内过量ROS的清除作用是否与rhNgb对细胞的毒性损伤有关,我们还采用CCK-8法对rhNgb的PC12细胞毒性进行了测定。结果发现,rhNgb具有一定的清除PC12细胞内过量ROS的能力,且没有细胞毒性。进一步,通过对不同物种的Ngb蛋白和mRNA序列进行生物信息学分析,结合已有的文献报道,发现组氨酸(histidine, His)在Ngb蛋白功能中起重要作用,因而推测His是Ngb蛋白中的关键氨基酸,并在此基础上使用引物突变法分别构建了突变体Ngb的原核表达载体(His64和His96单突变原核表达载体pBV220-Ngb(H64V)、pBV220-Ngb(H96A)和His64/96双突变原核表达载体pBV220-Ngb(H64V/H96A)),通过测序证明载体构建成功。同时,对突变体Ngb进行了原核表达,结果发现含突变体Ngb原核表达的菌体呈浅黄色(野生型Ngb原核表达产物为红色)。突变体的表达产物离心收集并超声裂解后,经SDS-PAGE和Western blotting分析可知,突变体Ngb能够正常表达,为研究上述关键氨基酸(His64、His96)在Ngb功能发挥中的作用提供了实验基础,并为后续研究Ngb清除自由基的作用机制奠定了基础。综上所述,本文在对rhNgb的抗氧化剂活性进行一系列研究的基础上,证明rhNgb是一种潜在的抗氧化剂,具有抗氧化能力,能够直接清除自由基,并能够清除细胞内过量的ROS,同时还证明rhNgb对Fe(II)具有螯合能力,对自由基造成的DNA损伤有一定的保护作用,而且其抗氧化能力和清除自由基活性可能是抗氧化损伤的关键所在,为进一步揭示Ngb的功能并向临床应用过渡提供了重要的实验依据。Ngb序列分析以及系列Ngb突变体表达载体的成功构建和表达,对于后续研究组氨酸在Ngb中的作用提供了物质保障。
[Abstract]:Free radicals are intermediates in the metabolism of the body, the free radicals in the human body physiological state is always in the dynamic balance of production and elimination process, and this balance mainly depends on the antioxidant system in order to maintain. However, excessive free radicals are important factors to produce oxidative stress, more easily in the body and all large biological molecules react and cause oxidative damage to cells and tissues. The important reason of free radical in the body too much and many diseases, such as neurodegenerative diseases, stroke, cancer and so on, so the removal of excessive free radicals has become an important strategy for prevention and treatment of this disease. Neuroglobin (neuroglobin, Ngb) is Burmester in 2000 found third kinds of oxygen carrying globin, mainly expressed in nervous system, with a high affinity for oxygen, can promote oxygen transfer to the mitochondria, improve the fabric of oxygen utilization in brain group Rate, may act as an endogenous neuroprotective factor against ischemia / hypoxia injury. Many studies have found that the oxygen carrying globin Ngb and the removal of excess free radicals, which indicates that Ngb may play an important role in the removal of excessive free radicals, but most reported by transfection of eukaryotic cell and transgenic technology means that Ngb can remove harmful free radical, protect cells and tissues against free radical damage. So, whether the Ngb protein has a direct free radical scavenging effect and its mechanism? This problem has not yet been clearly answered. This paper intends to focus on.
First of all, the study of determination of free radical scavenging activity of antioxidants in vitro and in cells, the recombinant human neuroglobin (recombinant human, neuroglobin, rhNgb) antioxidant, free radical scavenging activity, metal chelating ability and determination to DNA damage protection. Considering Ngb as a member of oxygen carrying beads a family of proteins, the antioxidant capacity is a more specific function, we use ABTS method and potassium ferricyanide reduction method of rhNgb oxidation and reduction ability were determined. The results showed that rhNgb has certain antioxidant capacity, but its antioxidant ability is lower than conventional antioxidants Vc, GSH and NAC; and reducing power determination results rhNgb showed that almost no reduction ability, which may be related to the oxidative state of rhNgb reduction.
The oxygen free radical scavenging activity of antioxidants is an important indicator of its antioxidant properties, so the study of rhNgb oxidation and reduction ability at the same time, the superoxide anion, hydrogen peroxide and hydroxyl radical were determined in the experiment. The rhNgb of scavenging superoxide anion, hydrogen peroxide, hydroxyl radical activity was detected respectively by four with nitro blue (nitro blue tetrazolium, NBT) - photoreduction method, H2O2 inactivated enzyme method, two adjacent Orthophenanthroline -Fe (II) colorimetric method. The results show that rhNgb has clear superoxide anion, hydrogen peroxide, hydroxyl radical activity. Further analysis shows that rhNgb has a very strong scavenging of superoxide anion, the activity of Vc is similar to rhNgb; scavenging activity of hydrogen peroxide is also strong, but its ability to scavenge hydroxyl radicals is weak, compared with Vc and NAC have a certain gap. In addition, we also rhNgb peroxide The activity of enzyme and the ability to scavenging DPPH free radical were detected. The results showed that rhNgb had little peroxidase activity and no ability to remove DPPH free radicals.
In the proof of rhNgb with the free radical scavenging activity at the same time, in order to better study the Ngb activity in the anti free radical injury, we also rhNgb Fe (II) chelating ability and the protective effect on DNA damage were discussed. The transition metal is easy to occur Fenton reaction in the body, produce hydroxyl free the base, so a metal chelating ability of antioxidants also reflect its antioxidant ability. The results showed that rhNgb of Fe transition metal (II) chelating ability with certain concentration, but its chelating ability is not entirely dependent on the low concentration of rhNgb, the metal chelating ability of iron ions increases with the concentration of rhNgb increased, when rhNgb reaches a certain concentration, the chelating ability began to decline. There is some relationship between the iron porphyrin ring which may exist in the rhNgb.
In order to better understand whether rhNgb of intracellular free radical scavenging activity also has, we rhNgb on cytotoxicity and cell clearance of excess ROS (reactive oxygen species, ROS) were detected. The intracellular ROS detection method has a lot of, starting from the angle of sensitivity, the clearance of rhNgb in PC12 cells in the excess capacity of ROS DCFH-DA fluorescent probe method we choose high sensitivity. At the same time, in order to determine whether the removal effect of rhNgb on PC12 cells in excess of ROS toxic damage to cells associated with rhNgb, we also collected were determined by PC12 to rhNgb cytotoxicity by CCK-8 method. The results showed that rhNgb has certain scavenging capacity PC12 cells of excessive ROS, and no cytotoxicity.
Further, the biological information of Ngb mRNA and protein sequences of different species analysis, combined with the existing literature, found that the histidine (histidine, His) play an important role in the function of Ngb protein, suggesting that His is a key amino acid in the Ngb protein, and on the basis of using the primers were constructed for prokaryotic mutation method the expression vector of Ngb mutant (His64 and His96 single mutant prokaryotic expression vector pBV220-Ngb (H64V), pBV220-Ngb (H96A) and His64/96 double mutant prokaryotic expression vector pBV220-Ngb (H64V/H96A)), by sequencing proved that carrier successfully constructed. At the same time, the mutants of Ngb by prokaryotic expression and found containing mutant Ngb prokaryotic the expression was pale yellow (wild type Ngb prokaryotic expression product is red.) expression of mutant product collected by centrifugation and after sonication, by SDS-PAGE and Western blotting analysis showed that the mutant Ngb It is a normal expression. It provides experimental basis for studying the role of His64 (His96) in Ngb function, and lays a foundation for subsequent research on the mechanism of Ngb scavenging free radicals.
In summary, this paper conducts a series of research on the antioxidant activity of rhNgb, that rhNgb is a potent antioxidant, antioxidant ability, capable of directly scavenging free radicals, and can remove the excessive ROS in cells, and that of rhNgb Fe (II) with chelating ability, the protective effect of DNA the damage of free radicals, and the antioxidant capacity and free radical scavenging activity may be the key to oxidative damage. In order to further reveal the function of Ngb and provide an important experimental basis for.Ngb sequence analysis and Ngb mutant expression vector is successfully constructed and the expression to clinical application, provides material guarantee for the follow-up study of histidine in Ngb.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

【参考文献】