發(fā)布時(shí)間：2018-05-13 07:27

  本文選題：5-HMF + 抗氧化損傷�。� 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2009年碩士論文

【摘要】： 氧化應(yīng)激與過氧化物積聚是導(dǎo)致多種疾病(如腦缺血、Alzheimer’s病(AD)、腦動(dòng)脈粥樣硬化等)的根本原因。H_2O_2是氧化應(yīng)激反應(yīng)密切相關(guān)的活性氧之一,其主要產(chǎn)物OH-具有很強(qiáng)的氧化性并可自由進(jìn)入細(xì)胞,破壞細(xì)胞內(nèi)活性氧和抗氧化代謝的平衡,使抗氧化酶等活性下降,同時(shí)會(huì)損傷體內(nèi)脂質(zhì)、蛋白質(zhì)和DNA等生物大分子,造成細(xì)胞內(nèi)酶活力變化、代謝紊亂、DNA損傷和細(xì)胞調(diào)亡。機(jī)體自身具有清除或修復(fù)氧化損傷的能力,例如超氧化物歧化酶(SOD)和過氧化物酶(POD)是機(jī)體內(nèi)重要的抗氧化酶系之一,其作用在于清除體內(nèi)的自由基;無嘌呤/無嘧啶核酸內(nèi)切酶(APE/Ref-1, apurinic/apyrimidinic endonuclease/redox factor-1)則在堿基切除修復(fù)(BER, base excise repair)中起關(guān)鍵作用,是參與DNA氧化損傷修復(fù)的重要抗氧化酶。氧化損傷時(shí)這些酶的表達(dá)及活性降低,因此,提高機(jī)體氧化損傷時(shí)抗氧化酶的表達(dá)/活性將有利于對(duì)上述疾病的預(yù)防及治療。 APE/Ref-1是個(gè)具有多種生物學(xué)功能的蛋白質(zhì),在許多生物過程中均發(fā)揮著重要作用。APE/Ref-1蛋白由318個(gè)氨基酸組成,其C末端功能區(qū)具有核酸內(nèi)切酶活性,當(dāng)DNA分子受氧化損傷時(shí),APE/Ref-1發(fā)揮核酸內(nèi)切酶活性參與DNA的堿基切除修復(fù):由糖基化酶(OGG1)移除受損堿基后,APE/Ref-1在無嘌呤/無嘧啶(apurinic/apyrimidinic, AP)部位的5′端切斷DNA鏈,DNA聚合酶以其外切酶作用切除AP部位的核苷酸,并根據(jù)堿基互補(bǔ)原則插入新的核苷酸進(jìn)行修復(fù);APE/Ref-1蛋白N末端具氧化還原功能,在眾多轉(zhuǎn)錄因子的激活中起重要作用,如維持Fos、Jun、HIF-1等轉(zhuǎn)錄因子的DNA結(jié)合域內(nèi)半胱氨酸處于還原狀態(tài),促進(jìn)轉(zhuǎn)錄因子的DNA結(jié)合功能。 APE/Ref-1對(duì)生物體的存活起著至關(guān)重要的作用,APE/Ref-1敲除小鼠在胚胎期E3.5時(shí)即發(fā)生死亡,APE/Ref-1的缺失使培養(yǎng)的細(xì)胞也難以存活。以上提示APE/Ref-1表達(dá)降低是細(xì)胞氧化損傷以至死亡的共同通路。因此,我們有理由相信以APE/Ref-1為靶點(diǎn),定向增加其蛋白表達(dá),或抑制其在氧化損傷過程中表達(dá)下調(diào)來防治氧化損傷性疾病的藥物研究將具有重要的科學(xué)意義及應(yīng)用價(jià)值。 5-羥甲基糠醛(5-hydroxymethyl-2-furaldehyde, 5-HMF)是廣泛存在含糖類植物中的一種小分子單體化合物,主要由己糖經(jīng)加熱分解產(chǎn)生。近年來研究表明5-HMF具有抗氧化、改善血液流變學(xué)等對(duì)人體有利的作用。本實(shí)驗(yàn)室最近研究發(fā)現(xiàn)5-HMF能延長(zhǎng)小鼠在嚴(yán)重低氧條件下的存活時(shí)間,提高細(xì)胞存活率,但其抗氧化損傷作用及機(jī)制尚不明確。本實(shí)驗(yàn)用H_2O_2誘導(dǎo)PC12細(xì)胞氧化損傷,研究5-HMF的抗氧化損傷作用及其作用機(jī)制。研究?jī)?nèi)容主要包括兩大部分:(1)5-HMF的抗氧化損傷作用;(2)5-HMF的抗氧化損傷作用機(jī)制的初步探討。主要研究APE/Ref-1是否參與了5-HMF的抗氧化作用。實(shí)驗(yàn)結(jié)果如下: 1. 5-HMF抗氧化損傷作用研究 以不同濃度的5-HMF處理細(xì)胞,在不同的時(shí)間點(diǎn)檢測(cè)細(xì)胞存活率,尋找到合適濃度的5-HMF。再用此濃度的5-HMF預(yù)處理PC12細(xì)胞,以H_2O_2處理細(xì)胞后檢測(cè)細(xì)胞存活,細(xì)胞形態(tài)學(xué)變化及DNA氧化損傷情況,研究發(fā)現(xiàn)5-HMF具有明顯的抗H_2O_2引起的氧化損傷作用。 1.1細(xì)胞存活檢測(cè) 1.1.1不同濃度5-HMF對(duì)細(xì)胞存活率的影響 用50μg/ml、100μg/ml、200μg /ml、400μg /ml的5-HMF分別處理PC12細(xì)胞2小時(shí)、6小時(shí)和24小時(shí)后,用MTT檢測(cè)細(xì)胞存活率,發(fā)現(xiàn)200μg /ml、400μg /ml的5-HMF作用6小時(shí)和24小時(shí)后,細(xì)胞存活率明顯下降,而50μg/ml、100μg/ml的5-HMF在作用24小時(shí)后細(xì)胞存活率仍未受影響,因此選用這兩個(gè)濃度的5-HMF,研究其在抗氧化損傷中的作用。 1.1.2 H_2O_2對(duì)細(xì)胞存活的影響及5-HMF的作用 分別用50μg/ml、100μg/ml 5-HMF預(yù)處理細(xì)胞30分鐘后,再用400μM H_2O_2處理1小時(shí),MTT檢測(cè)細(xì)胞存活率,結(jié)果表明H_2O_2處理后細(xì)胞存活率降低,50μg/ml和100μg/ml 5-HMF則能明顯提高H_2O_2氧化損傷后的細(xì)胞存活率。 1.2細(xì)胞形態(tài)學(xué)觀察 進(jìn)一步用倒置相差顯微鏡觀察細(xì)胞形態(tài)學(xué)上的變化,發(fā)現(xiàn)400μM H_2O_2處理后細(xì)胞皺縮變小,細(xì)胞數(shù)目明顯減少,50μg/ml和100μg/ml 5-HMF預(yù)處理后能減緩H_2O_2引發(fā)的形態(tài)學(xué)改變。 1.3 DNA氧化損傷檢測(cè) 為確認(rèn)H_2O_2引起的DNA氧化損傷以及5-HMF的作用,實(shí)驗(yàn)采用免疫細(xì)胞化學(xué)方法檢測(cè)DNA氧化損傷標(biāo)志物8-oxo-dG的表達(dá)變化情況。結(jié)果發(fā)現(xiàn),400μM H_2O_2處理PC12細(xì)胞1小時(shí)后8-oxo-dG表達(dá)增強(qiáng),而50μg/ml和100μg/ml 5-HMF明顯抑制了H_2O_2對(duì)8-oxo-dG的誘導(dǎo)作用,該結(jié)果表明5-HMF具有抗H_2O_2引起的DNA氧化損傷作用。 2. 5-HMF抗氧化損傷作用機(jī)制的初步研究 由于APE/Ref-1在DNA氧化損傷的堿基切除修復(fù)中具有重要作用,它很有可能是5-HMF抗氧化作用的靶點(diǎn),為此,我們著重探討了APE/Ref-1在5-HMF抗氧化損傷中的作用。 2.1 5-HMF對(duì)APE/Ref-1表達(dá)變化的影響 2.1.1 5-HMF對(duì)APE/Ref-1蛋白表達(dá)的影響 免疫細(xì)胞化學(xué)結(jié)果顯示:400μM H_2O_2導(dǎo)致APE/Ref-1表達(dá)明顯下調(diào),5-HMF處理本身并未增加APE/Ref-1表達(dá),但卻抑制了APE/Ref-1在H_2O_2處理后的表達(dá)下調(diào)。為確認(rèn)5-HMF對(duì)APE/Ref-1蛋白表達(dá)的影響,實(shí)驗(yàn)進(jìn)一步用Western blot方法檢驗(yàn)了APE/Ref-1的表達(dá)情況,經(jīng)驗(yàn)證與免疫細(xì)胞化學(xué)檢測(cè)的結(jié)果一致。 2.1.2 5-HMF對(duì)APE/Ref-1 mRNA表達(dá)的影響 5-HMF對(duì)APE/Ref-1蛋白表達(dá)的影響是否與其對(duì)APE/Ref-1 mRNA水平的調(diào)控有關(guān)呢?為此我們以RT-PCR檢測(cè)了APE/Ref-1 mRNA表達(dá)情況,結(jié)果顯示H_2O_2處理未改變APE/Ref-1 mRNA水平,亦不受5-HMF的影響。 以上實(shí)驗(yàn)表明,5-HMF能抑制H_2O_2引起的APE/Ref-1蛋白表達(dá)下調(diào),且與其對(duì)APE/Ref-1的轉(zhuǎn)錄調(diào)節(jié)無關(guān)。此外,APE/Ref-1作為核酸內(nèi)切酶,其活性是否受參與5-HMF的抗氧化損傷作用尚不清楚,因此接下來我們采用APE/Ref-1抑制劑MX抑制其酶活性后,進(jìn)一步檢測(cè)了5-HMF的抗氧化損傷作用。 2.2 APE/Ref-1酶活性介導(dǎo)了5-HMF的抗氧化損傷作用 2.2 .1檢測(cè)不同濃度MX在不同時(shí)間點(diǎn)對(duì)PC12細(xì)胞存活率的影響 以1 mM、2.5 mM、5 mM、7.5 mM和10 mM MX分別處理PC12細(xì)胞2小時(shí)、16小時(shí)和24小時(shí),MTT檢測(cè)細(xì)胞存活率,發(fā)現(xiàn)7.5 mM和10 mM MX作用2小時(shí)細(xì)胞生長(zhǎng)受到抑制,而此時(shí)1 mM、2.5 mM和5 mM MX對(duì)細(xì)胞存活基本沒有影響,結(jié)合文獻(xiàn)報(bào)道我們最后選用5 mM MX抑制APE/Ref-1酶活性。 2.2.2 MX處理后對(duì)5-HMF抗氧化損傷作用的影響 用5 mM MX抑制APE/Ref-1核酸內(nèi)切酶活性,檢測(cè)5-HMF的抗氧化損傷作用。結(jié)果顯示,APE/Ref-1酶活性受抑制后,5-HMF原有的提高氧化損傷時(shí)細(xì)胞存活能力降低,并且其抗DNA氧化損傷作用也被減弱。這些結(jié)果暗示5-HMF的抗氧化損傷作用是通過APE/Ref-1的酶活性來介導(dǎo)的。 綜上所述,5-HMF具有明顯抗H_2O_2引起的氧化損傷作用,且其作用是通過穩(wěn)定APE/Ref-1蛋白表達(dá),使其發(fā)揮核酸內(nèi)切酶活性來實(shí)現(xiàn)的。5-HMF如何抑制APE/Ref-1在氧化損傷時(shí)的降解,以及APE/Ref-1的氧化還原活性-即對(duì)轉(zhuǎn)錄因子的調(diào)節(jié)功能是否參與了5-HMF的抗氧化損傷作用還需進(jìn)一步深入研究。
[Abstract]:Oxidative stress and peroxide accumulation are the fundamental causes of a variety of diseases (such as cerebral ischemia, Alzheimer 's disease (AD), and cerebral atherosclerosis)..H_2O_2 is one of the reactive oxygen species closely related to oxidative stress. Its main product, OH-, is highly oxidizing and can be derived from the cells and destroys intracellular reactive oxygen species and antioxidant metabolism. The balance, which reduces the activity of antioxidant enzymes, and damages biological macromolecules such as lipid, protein and DNA in the body, causes changes in enzyme activity, metabolic disorder, DNA damage and cell death. The body itself has the ability to remove or repair oxidative damage, such as superoxide dismutase (SOD) and peroxidase (POD), which are internal weight of the body. One of the antioxidant enzymes required is to scavenge free radicals, and purine / apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1, endonuclease/redox factor-1) plays a key role in base excision repair (BER, base excise repair), and is an important antioxidant involved in the repair of DNA oxidative damage. The expression and activity of these enzymes decrease. Therefore, improving the expression and activity of antioxidant enzymes in oxidative damage of the body will be beneficial to the prevention and treatment of these diseases.
APE/Ref-1 is a protein with many biological functions and plays an important role in many biological processes..APE/Ref-1 protein is composed of 318 amino acids. Its C terminal functional region has endonuclease activity. When DNA molecules are damaged by oxidation, APE/Ref-1 plays the activity of endonuclease in nucleic acid radical resection and repair of DNA, which is derived from glycosyl group. After the enzyme (OGG1) removal of the damaged base, APE/Ref-1 cuts the DNA chain at the 5 'end of the purine / pyrimidine (apurinic/apyrimidinic, AP) site. The DNA polymerase excuses the nucleotide of the AP site by its external enzyme action, and inserts the new nucleotide according to the principle of base complementarity. The N terminal of APE/Ref-1 protein has the redox function, in many turns. The activation of transcription factors plays an important role, such as maintaining the reduction state of cysteine in the DNA binding domain of the transcription factors such as Fos, Jun, HIF-1 and other transcription factors, and promoting the DNA binding function of the transcription factors.
APE/Ref-1 plays a vital role in the survival of the organism. APE/Ref-1 knocks off mice at the embryonic stage of E3.5, and the absence of APE/Ref-1 makes the cultured cells difficult to survive. These suggest that the decrease in APE/Ref-1 expression is the common pathway of cell oxidative damage and even death. For this reason, we have reason to believe that APE/Ref-1 is the target, It is of great scientific significance and application value to increase the expression of its protein or inhibit its regulation of down regulation in the process of oxidative damage to prevent oxidative damage.
5- hydroxymethyl furfural (5-hydroxymethyl-2-furaldehyde, 5-HMF) is a kind of small molecular monomer compound widely existed in sugar containing plants, which is mainly decomposed by hexose. In recent years, studies have shown that 5-HMF has antioxidation and improves blood rheology and other beneficial effects on human body. Recently, the study found that 5-HMF can prolong the small size of 5-HMF. The survival time of rat under severe hypoxic conditions increased the survival rate of cells, but the effect and mechanism of antioxidant damage were not clear. This experiment used H_2O_2 to induce oxidative damage in PC12 cells and study the antioxidant effects and mechanism of 5-HMF. The main contents of the study include two parts: (1) the antioxidant effect of 5-HMF; (2) the resistance of 5-HMF. A preliminary study on the mechanism of oxidative damage. We mainly studied whether APE/Ref-1 was involved in the antioxidant effect of 5-HMF.
Study on the effect of 1. 5-HMF on antioxidant injury
The cells were treated with different concentrations of 5-HMF, and the cell survival rate was detected at different time points. The suitable concentration of 5-HMF. was found and then the PC12 cells were pretreated with this concentration of 5-HMF, and the cells survived, the cell morphology and the DNA oxidative damage were detected after H_2O_2 treatment. It was found that 5-HMF has obvious oxidation resistance to H_2O_2. Damage effect.
1.1 cell survival detection
The effect of different concentrations of 5-HMF on cell survival rate of 1.1.1
After 2 hours, 6 hours and 24 hours of PC12 cells treated with 50 mu g/ml, 100 mu g/ml, 200 mu g /ml and 400 mu g /ml, the cell survival rate was detected by MTT, and 200 micron g /ml. After 6 hours and 24 hours of 6 hours and 24 hours, the survival rate of cell viability was still not affected by 50 mu. Therefore, we chose these two concentrations of 5-HMF to study its role in antioxidant damage.
The effect of 1.1.2 H_2O_2 on cell survival and the role of 5-HMF
The cells were pretreated with 50 g/ml and 100 g/ml 5-HMF for 30 minutes respectively, then 400 M H_2O_2 was treated for 1 hours. The survival rate of cells was detected by MTT. The results showed that the survival rate of cells decreased after H_2O_2 treatment, and the survival rate of cells after H_2O_2 oxidative damage was obviously improved.
Morphological observation of 1.2 cells
The morphological changes of cells were observed by inverted phase contrast microscope. It was found that the cell shrinkage was smaller and the number of cells decreased significantly after 400 M H_2O_2 treatment. The morphological changes caused by H_2O_2 could be slowed down after 50 g/ml and 100 mu g/ml 5-HMF pretreatment.
1.3 DNA oxidative damage detection
In order to confirm the oxidative damage of DNA caused by H_2O_2 and the effect of 5-HMF, the expression of 8-oxo-dG was detected by immunocytochemistry. The results showed that the expression of 8-oxo-dG was enhanced after 1 hours of PC12 cells treated with 400 u M H_2O_2, while 50 mu g/ml and 100 mu g /ml significantly inhibited the induction of PC12. The results indicate that 5-HMF has anti H_2O_2 induced oxidative damage to DNA.
Preliminary study on the mechanism of anti oxidative damage of 2. 5-HMF
As APE/Ref-1 plays an important role in the alkali radical resection and repair of DNA oxidative damage, it is likely to be the target of the antioxidant activity of 5-HMF. Therefore, we have focused on the role of APE/Ref-1 in the oxidative damage of 5-HMF.
The effect of 2.1 5-HMF on the changes of APE/Ref-1 expression
The effect of 2.1.1 5-HMF on the expression of APE/Ref-1 protein
The immunocytochemical results showed that the expression of APE/Ref-1 was obviously downregulated by 400 M H_2O_2, and the 5-HMF treatment itself did not increase the expression of APE/Ref-1, but it inhibited the downregulation of APE/Ref-1 expression after H_2O_2 treatment. To confirm the effect of 5-HMF on the expression of APE/Ref-1 protein, the experiment further tested the expression of APE/Ref-1 by Western blot method. The results were consistent with the results of immunocytochemistry.
The effect of 2.1.2 5-HMF on the expression of APE/Ref-1 mRNA
Is the effect of 5-HMF on the expression of APE/Ref-1 protein related to the regulation of the level of APE/Ref-1 mRNA? For this reason, we detected the expression of APE/Ref-1 mRNA by RT-PCR. The results showed that H_2O_2 treatment did not change the APE/Ref-1 mRNA level and was not affected by 5-HMF.
The above experiments show that 5-HMF can inhibit the downregulation of APE/Ref-1 protein expression caused by H_2O_2 and is not related to the transcriptional regulation of APE/Ref-1. In addition, it is not clear whether APE/Ref-1 acts as an endonuclease, and its activity is not known to be involved in the antioxidant damage of 5-HMF, so we then use the APE/Ref-1 inhibitor MX to inhibit its enzyme activity. The anti oxidative damage effect of 5-HMF was detected step by step.
2.2 APE/Ref-1 enzyme activity mediates the antioxidant damage of 5-HMF.
2.2.1 detection of different concentrations of MX at different time points on the survival rate of PC12 cells.
With 1 mM, 2.5 mM, 5 mM, 7.5 mM and 10 mM MX respectively, PC12 cells were treated for 2 hours, 16 hours and 24 hours, MTT detected cell viability, and the growth of 7.5 mM and 10 mM MX was inhibited in 2 hours. Activity.
Effect of 2.2.2 MX treatment on antioxidant damage of 5-HMF
The inhibitory activity of APE/Ref-1 endonuclease with 5 mM MX was used to detect the antioxidant effect of 5-HMF. The results showed that after the activity of APE/Ref-1 enzyme was inhibited, the survival ability of 5-HMF was reduced when the oxidation damage was raised, and the anti DNA oxidative damage was also weakened. These results suggest that the antioxidant effect of 5-HMF is through APE/Ref-1. The enzyme activity is mediated.
To sum up, 5-HMF has a significant effect on oxidative damage induced by H_2O_2, and its effect is by stabilizing the expression of APE/Ref-1 protein to make the.5-HMF inhibit the degradation of APE/Ref-1 during oxidative damage and the redox activity of APE/Ref-1, namely, whether the regulatory function of the transcription factor is involved. The antioxidant effect of 5-HMF needs further study.

【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】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R363

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本文編號(hào)：1882235