發(fā)布時(shí)間：2018-07-23 10:49

【摘要】：目的：膠霉毒素（GT）廣泛存在于里氏木霉、青霉、煙曲霉和白色念珠菌等致病菌或環(huán)境微生物的次生代謝產(chǎn)物中，研究證明GT的產(chǎn)生主要來(lái)源于煙曲霉毒素，，另外通過(guò)液相色譜串聯(lián)質(zhì)譜（HPLC）的方法檢測(cè)發(fā)現(xiàn)膠霉毒素存在于動(dòng)物（如豬，奶牛，家禽）的飼料中，也產(chǎn)生于玉米的貯存過(guò)程。體外實(shí)驗(yàn)表明膠霉毒素有很強(qiáng)的抗菌、抗病毒作用,呈現(xiàn)出較好的藥用前景，曾一時(shí)被認(rèn)為可用于抗腫瘤治療。但隨后的研究表明膠霉毒素具有抑制血管生成、免疫抑制，抑制NF-kB及其他特定基因表達(dá)及介導(dǎo)細(xì)胞凋亡等毒性。本研究通過(guò)觀察膠霉毒素誘導(dǎo)HEK-293細(xì)胞的遺傳毒性，以及其引起ROS的產(chǎn)生和溶酶體膜通透的改變，旨在探討GT對(duì)DNA損傷的可能毒性機(jī)制，為進(jìn)一步評(píng)估膠霉毒素對(duì)人類的健康影響提供試驗(yàn)及理論依據(jù)。 方法：以HEK-293細(xì)胞作為試驗(yàn)系統(tǒng)。本實(shí)驗(yàn)運(yùn)用單細(xì)胞凝膠電泳試驗(yàn)（SCGE）和蛋白質(zhì)印跡法（western blot）檢測(cè)GT對(duì)體外HEK-293細(xì)胞DNA損傷情況以及DNA損傷蛋白的高表達(dá)（ATM和P53)水平，進(jìn)而驗(yàn)證膠霉毒素的DNA損傷毒性。為了進(jìn)一步探討其可能的DNA損傷機(jī)制，以2’,7’—二氫二氯熒光素（DCFH）和吖啶橙（AO）分別測(cè)定膠霉毒素對(duì)細(xì)胞內(nèi)活性氧（ROS）以及溶酶體膜穩(wěn)定性的影響，通過(guò)N-乙酰半胱氨酸（NAC）干預(yù)試驗(yàn)觀察氧化性損傷在DNA損傷中的作用，以及對(duì)溶酶體膜穩(wěn)定性的影響。試驗(yàn)結(jié)果用SPSS v12.7統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析。 結(jié)果：當(dāng)膠霉毒素（GT)作用于HEK-293細(xì)胞1小時(shí)后，彗星試驗(yàn)顯示其引起DNA雙鏈斷裂，熒光顯微鏡下與DMSO空白對(duì)照組相比，細(xì)胞成彗星樣拖尾，其趨勢(shì)隨著濃度而遞增，其中尾長(zhǎng)增長(zhǎng)，尾DNA%以及DNA%含量明顯增大；同時(shí)，GT（0.3125μM～1.25μM）可導(dǎo)致細(xì)胞內(nèi)ROS的表達(dá)水平升高，溶酶體膜穩(wěn)定性下降。用NAC干預(yù)劑預(yù)處理后，GT所引起的HEK293細(xì)胞的拖尾現(xiàn)象、細(xì)胞內(nèi)ROS以及溶酶體膜通透都有明顯地減弱。作用24小時(shí)后，膠霉毒素同樣能誘導(dǎo)HEK293細(xì)胞的DNA損傷，其損傷蛋白的表達(dá)水平呈劑量依賴效應(yīng)趨勢(shì)，而此時(shí)未出現(xiàn)HEK293細(xì)胞的凋亡發(fā)生，DNA損傷蛋白ATM/P53的表達(dá)水平可被抗氧化劑NAC有效干預(yù)，結(jié)果出現(xiàn)DNA損傷高表達(dá)蛋白明顯的下降趨勢(shì)。 結(jié)論：膠霉毒素在高濃度0.625μM-1.25μM的作用下能夠引起HEK293細(xì)胞的DNA損傷，說(shuō)明其具有遺傳毒性。同時(shí)，GT可導(dǎo)致HEK-293細(xì)胞內(nèi)ROS生成增加，使細(xì)胞處于氧化應(yīng)激狀態(tài)，并使溶酶體膜穩(wěn)定性下降。而抗氧化劑NAC在降低了細(xì)胞內(nèi)ROS水平的同時(shí)，對(duì)溶酶體膜穩(wěn)定性呈現(xiàn)保護(hù)趨勢(shì)，同樣條件下減弱了GT誘導(dǎo)的DNA鏈斷裂程度，這些結(jié)果表明膠霉毒素能引起HEK-293細(xì)胞內(nèi)溶酶體膜穩(wěn)定性下降及DNA損傷，其作用機(jī)制與氧化應(yīng)激性有關(guān)。DNA損傷蛋白（ATM，P53）的高表達(dá)以及NAC對(duì)其的保護(hù)作用，進(jìn)一步說(shuō)明了膠霉毒素能引起氧化性DNA損傷。
[Abstract]:Objective: collotoxin (GT) is widely found in the secondary metabolites of pathogenic bacteria, such as Trichoderma ribergii, Penicillium, Aspergillus fumigatus and Candida albicans. In addition, it was found by liquid chromatography-tandem mass spectrometry (HPLC) that collotoxin exists in the feed of animals (such as pigs, cows and poultry) and also occurs in the storage process of maize. In vitro experiments showed that collotoxin had strong antimicrobial and antiviral effects and showed a good medicinal prospect, which had been considered as an antitumor therapy for some time. However, subsequent studies showed that collotoxin could inhibit angiogenesis, immunosuppression, inhibit the expression of NF-kB and other specific genes, and mediate apoptosis. The aim of this study was to investigate the possible toxic mechanism of GT on DNA damage by observing the genotoxicity of HEK-293 cells induced by collotoxin, the production of Ros and the changes of lysosomal membrane permeability. To provide experimental and theoretical basis for further evaluation of the effects of collotoxin on human health. Methods: HEK-293 cells were used as test system. In this study, single cell gel electrophoresis (SCGE) and Western blotting (western blot) were used to detect the DNA damage of HEK-293 cells and the high expression of DNA damage protein (ATM and p53). In order to further study the possible DNA damage mechanism, the effects of collotoxin on intracellular reactive oxygen species (Ros) and lysosomal membrane stability were determined by using 2H7 Dichlorofluorescein (DCFH) and acridine orange (AO), respectively. The effects of oxidative damage on DNA damage and the stability of lysosomal membrane were observed by N-acetylcysteine (NAC) intervention. The results were analyzed by SPSS v12.7 software. Results: when gelatoxin (GT) was treated on HEK-293 cells for 1 hour, comet assay showed that it caused DNA double strand break. Compared with DMSO blank control group, the cells formed comet-like tail under fluorescence microscope, and the trend increased with the concentration. The increase of tail length, the increase of tail DNA% and the increase of DNA%, and the increase of Ros expression and the decrease of lysosomal membrane stability. After pretreatment with NAC, the tail trailing phenomenon, intracellular Ros and lysosomal membrane permeability of HEK293 cells induced by glutamate GT were significantly decreased. After exposure for 24 hours, collotoxin could also induce DNA damage in HEK293 cells, and the expression level of the damage protein was in a dose-dependent manner. However, no apoptotic DNA damage protein ATM / P 53 was detected in HEK293 cells, which could be effectively interfered by antioxidant NAC, resulting in a decrease of DNA damage overexpression protein. Conclusion: collotoxin can induce DNA damage in HEK293 cells at high concentration of 0.625 渭 M-1.25 渭 M, indicating its genotoxicity. At the same time, GT could increase Ros production in HEK-293 cells, make the cells under oxidative stress and decrease the stability of lysosomal membrane. However, the antioxidant NAC not only decreased the intracellular Ros level, but also protected the stability of lysosomal membrane. At the same time, the degree of DNA strand break induced by GT was weakened under the same conditions. These results suggest that collotoxin can induce the degradation of lysosomal membrane stability and DNA damage in HEK-293 cells. The mechanism is related to the overexpression of ATMN p53 and the protective effect of NAC on it. It is further explained that collotoxin can cause oxidative DNA damage.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R114

【共引文獻(xiàn)】

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本文編號(hào)：2139153