本文選題：腦紅蛋白　切入點(diǎn)：抗氧化　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2010年碩士論文

【摘要】： 自由基是機(jī)體正常代謝的中間產(chǎn)物,在生理狀態(tài)下人體內(nèi)自由基始終處于產(chǎn)生與清除的動(dòng)態(tài)平衡過程中,而此平衡主要依靠機(jī)體內(nèi)的抗氧化系統(tǒng)來維持。然而,過量的自由基是產(chǎn)生氧化應(yīng)激的重要因素,在機(jī)體內(nèi)較易與各種生物大分子發(fā)生反應(yīng)而導(dǎo)致細(xì)胞和組織氧化損傷。機(jī)體內(nèi)過多的自由基同時(shí)還是許多疾病產(chǎn)生的重要原因,如神經(jīng)系統(tǒng)退行性疾病、腦中風(fēng)、腫瘤等,因此清除過量自由基已成為防治此類疾病的重要策略。腦紅蛋白(neuroglobin,Ngb)是Burmester于2000年新發(fā)現(xiàn)的第三類攜氧珠蛋白,主要表達(dá)于神經(jīng)系統(tǒng),與氧有很高的親和力,能促進(jìn)氧傳遞到線粒體,提高腦組織對(duì)氧的利用率,可作為內(nèi)源性神經(jīng)保護(hù)因子保護(hù)神經(jīng)元抵御缺血/缺氧性損傷。很多研究發(fā)現(xiàn),具有攜氧能力的珠蛋白Ngb與清除過量自由基有關(guān),這說明Ngb在清除過多自由基方面可能起重要作用,但目前多數(shù)報(bào)道是通過真核細(xì)胞轉(zhuǎn)染技術(shù)和轉(zhuǎn)基因等手段,證明Ngb能夠清除過量的有害自由基,保護(hù)細(xì)胞和組織抵抗自由基損傷。那么,Ngb蛋白是否直接具有清除自由基作用,其機(jī)理如何?這一問題尚未得到明確回答。本文擬對(duì)此進(jìn)行重點(diǎn)研究。 首先,利用體外和細(xì)胞內(nèi)測(cè)定抗氧化劑清除自由基活性的研究方法,對(duì)重組人源腦紅蛋白(recombinant human neuroglobin, rhNgb)的抗氧化能力、清除自由基活性、金屬螯合能力以及對(duì)DNA氧化損傷的保護(hù)作用進(jìn)行測(cè)定�？紤]到Ngb作為攜氧珠蛋白家族中的一員,其抗氧化能力是一個(gè)比較特定的功能,我們分別采用ABTS法和鐵氰化鉀還原法對(duì)rhNgb的抗氧化和還原能力進(jìn)行測(cè)定,結(jié)果發(fā)現(xiàn)rhNgb具有一定的抗氧化能力,但其抗氧化能力要低于常規(guī)抗氧化劑Vc、GSH以及NAC;而還原能力測(cè)定結(jié)果表明,rhNgb幾乎無還原能力,這可能與rhNgb本身的氧化還原狀態(tài)有關(guān)。 由于抗氧化劑清除氧自由基的活性是其抗氧化屬性的重要指標(biāo),因此在研究rhNgb抗氧化和還原能力的同時(shí),我們對(duì)其清除超氧陰離子、過氧化氫、羥自由基等進(jìn)行了測(cè)定。本實(shí)驗(yàn)中rhNgb清除超氧陰離子、過氧化氫、羥自由基活性的檢測(cè)分別采用四唑硝基藍(lán)(nitro blue tetrazolium,NBT)-光還原法、H2O2滅活酶法、鄰二氮菲-Fe(II)比色法進(jìn)行。結(jié)果表明,rhNgb具有明確的清除超氧陰離子、過氧化氫、羥自由基等活性。進(jìn)一步分析可知,rhNgb對(duì)超氧陰離子有很強(qiáng)的清除能力,其活性與Vc相差不大;rhNgb對(duì)過氧化氫的清除活性也較強(qiáng),但其清除羥自由基能力較弱,與Vc和NAC相比有一定差距。另外,我們還對(duì)rhNgb的過氧化物酶活性和清除DPPH自由基能力進(jìn)行了檢測(cè)。結(jié)果發(fā)現(xiàn),rhNgb幾乎沒有過氧化物酶活性,也沒有清除DPPH自由基的能力。 在證明rhNgb具有上述清除自由基活性的同時(shí),為了更好地研究Ngb在抗自由基損傷方面的其他活性,我們還對(duì)rhNgb的Fe(II)螯合能力和對(duì)DNA氧化損傷的保護(hù)作用進(jìn)行了探討。過渡金屬在機(jī)體內(nèi)易于發(fā)生Fenton反應(yīng),產(chǎn)生羥自由基,因此抗氧化劑的金屬螯合能力也是其抗氧化能力的一種體現(xiàn)。結(jié)果發(fā)現(xiàn),rhNgb對(duì)過渡金屬Fe(II)具有一定的螯合能力,但其螯合能力并非完全的濃度依賴性,在低濃度rhNgb時(shí),其金屬鐵離子的螯合能力隨著rhNgb濃度升高而增加,當(dāng)rhNgb達(dá)到一定濃度時(shí),其螯合能力開始下降。這可能與rhNgb中存在的鐵卟啉環(huán)有一定的關(guān)系。 為了更好地理解rhNgb是否對(duì)細(xì)胞內(nèi)的自由基也具有清除活性,我們對(duì)rhNgb的細(xì)胞毒性和清除細(xì)胞過量活性氧(reactive oxygen species,ROS)進(jìn)行了檢測(cè)。目前對(duì)細(xì)胞內(nèi)ROS檢測(cè)方法有很多,從靈敏度等角度出發(fā),我們選用靈敏度較高的DCFH-DA熒光探針法對(duì)rhNgb清除PC12細(xì)胞內(nèi)過量ROS能力進(jìn)行了研究。同時(shí),為了便于確定rhNgb對(duì)PC12細(xì)胞內(nèi)過量ROS的清除作用是否與rhNgb對(duì)細(xì)胞的毒性損傷有關(guān),我們還采用CCK-8法對(duì)rhNgb的PC12細(xì)胞毒性進(jìn)行了測(cè)定。結(jié)果發(fā)現(xiàn),rhNgb具有一定的清除PC12細(xì)胞內(nèi)過量ROS的能力,且沒有細(xì)胞毒性。 進(jìn)一步,通過對(duì)不同物種的Ngb蛋白和mRNA序列進(jìn)行生物信息學(xué)分析,結(jié)合已有的文獻(xiàn)報(bào)道,發(fā)現(xiàn)組氨酸(histidine, His)在Ngb蛋白功能中起重要作用,因而推測(cè)His是Ngb蛋白中的關(guān)鍵氨基酸,并在此基礎(chǔ)上使用引物突變法分別構(gòu)建了突變體Ngb的原核表達(dá)載體(His64和His96單突變?cè)吮磉_(dá)載體pBV220-Ngb(H64V)、pBV220-Ngb(H96A)和His64/96雙突變?cè)吮磉_(dá)載體pBV220-Ngb(H64V/H96A)),通過測(cè)序證明載體構(gòu)建成功。同時(shí),對(duì)突變體Ngb進(jìn)行了原核表達(dá),結(jié)果發(fā)現(xiàn)含突變體Ngb原核表達(dá)的菌體呈淺黃色(野生型Ngb原核表達(dá)產(chǎn)物為紅色)。突變體的表達(dá)產(chǎn)物離心收集并超聲裂解后,經(jīng)SDS-PAGE和Western blotting分析可知,突變體Ngb能夠正常表達(dá),為研究上述關(guān)鍵氨基酸(His64、His96)在Ngb功能發(fā)揮中的作用提供了實(shí)驗(yàn)基礎(chǔ),并為后續(xù)研究Ngb清除自由基的作用機(jī)制奠定了基礎(chǔ)。 綜上所述,本文在對(duì)rhNgb的抗氧化劑活性進(jìn)行一系列研究的基礎(chǔ)上,證明rhNgb是一種潛在的抗氧化劑,具有抗氧化能力,能夠直接清除自由基,并能夠清除細(xì)胞內(nèi)過量的ROS,同時(shí)還證明rhNgb對(duì)Fe(II)具有螯合能力,對(duì)自由基造成的DNA損傷有一定的保護(hù)作用,而且其抗氧化能力和清除自由基活性可能是抗氧化損傷的關(guān)鍵所在,為進(jìn)一步揭示Ngb的功能并向臨床應(yīng)用過渡提供了重要的實(shí)驗(yàn)依據(jù)。Ngb序列分析以及系列Ngb突變體表達(dá)載體的成功構(gòu)建和表達(dá),對(duì)于后續(xù)研究組氨酸在Ngb中的作用提供了物質(zhì)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.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R341

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 張智清,姚立紅,侯云德;含P_RP_L啟動(dòng)子的原核高效表達(dá)載體的組建及其應(yīng)用[J];病毒學(xué)報(bào);1990年02期

2 徐文琳,王春麗,張永亮,廖志勇,余利紅,孟凡偉,王新興,尹昭云,錢令嘉,張成崗;腦紅蛋白與Na~+,K~+-ATP酶β2亞基的相互作用及作用位點(diǎn)的鑒定[J];生物化學(xué)與生物物理學(xué)報(bào);2003年09期

3 方允中,楊勝,伍國耀;自由基穩(wěn)衡性動(dòng)態(tài)[J];生理科學(xué)進(jìn)展;2004年03期

4 吳永紅;王利紅;高艷;張成崗;;重組人源腦紅蛋白的高效表達(dá)、純化及鑒定[J];中國生物工程雜志;2009年09期

5 楊芬;張瑞萍;賀玖明;再帕爾·阿不力孜;;羥自由基的產(chǎn)生、捕集及檢測(cè)方法[J];藥學(xué)學(xué)報(bào);2007年07期

6 徐向榮,王文華,李華斌;羥自由基的測(cè)定方法[J];中國衛(wèi)生檢驗(yàn)雜志;1997年05期

相關(guān)博士學(xué)位論文 前1條

1 陳婷方;腦紅蛋白神經(jīng)保護(hù)功能及其機(jī)制的初步研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2008年

，

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