發(fā)布時(shí)間：2018-05-13 08:41

  本文選題：黃曲霉毒素B_1 + 氧化損傷�。� 參考：《西南大學(xué)》2017年碩士論文

【摘要】：黃曲霉毒素B_1(Aflatoxin B_1,AFB_1)是目前已知的真菌毒素中毒性最強(qiáng)、對(duì)人類威脅最大的一種毒素。由于其極強(qiáng)的致癌性,并且其化學(xué)結(jié)構(gòu)穩(wěn)定高溫下難以被破壞分解,AFB_1的暴露最終將會(huì)導(dǎo)致各類急性或亞慢性中毒的發(fā)生。雖然AFB_1的致癌作用及其相關(guān)機(jī)理已被證實(shí),但其對(duì)生物體造成的氧化性、遺傳性損傷及其相關(guān)機(jī)制的研究并不十分明確,這并不利于對(duì)黃曲霉毒素(Aflatoxins,AFT)的毒性機(jī)制的深入研究,同時(shí)也對(duì)AFB_1的防御體系建立造成了一定的阻礙。本實(shí)驗(yàn)對(duì)AFB_1產(chǎn)生的氧化性、遺傳性毒害作用及相關(guān)分子作用機(jī)制進(jìn)行了研究,為全面評(píng)估AFB_1的毒性同時(shí)為闡明其他類別的黃曲霉毒素的毒性機(jī)理提供一定的借鑒,同時(shí)也為黃曲霉毒素的降解脫毒或防御體內(nèi)中毒提供更多的參考。本實(shí)驗(yàn)首先選用40只雄性小鼠,在亞慢性條件下進(jìn)行連續(xù)50天3種不同劑量(0.09375、0.375、1.5mg/kg b.w./day)的AFB_1染毒,對(duì)血清及臟器中各氧化損傷指標(biāo)、肝細(xì)胞凋亡及其相關(guān)信號(hào)通路等進(jìn)行研究,以評(píng)估其造成的氧化性損傷及分子機(jī)制;對(duì)肝臟中DNA鏈斷裂、交聯(lián),細(xì)胞微核及精子畸形等指標(biāo)進(jìn)行考察,以評(píng)估其遺傳毒性及分子機(jī)制。并研究體外條件下AFB_1與DNA之間的相互作用機(jī)制及其與遺傳毒性的相關(guān)性。具體的結(jié)果如下:(1)經(jīng)過50天的AFB_1染毒后,AFB_1對(duì)小鼠機(jī)體能夠造成明顯的臟器損傷、血清中谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)水平的顯著性升高。各染毒組小鼠體重隨染毒劑量的增加而降低,且肝臟、腎臟、睪丸三種器官重量也出現(xiàn)下降趨勢,表明該三種臟器受到AFB_1染毒的影響。AFB_1對(duì)機(jī)體造成的病理性損傷具體為:低劑量時(shí)以炎癥效應(yīng)為主,高劑量時(shí)則導(dǎo)致肝細(xì)胞局部壞死、腎小球腫脹及腎小管上皮細(xì)胞壞死等情況。(2)亞慢性條件下,AFB_1能夠?qū)π∈蟾闻K產(chǎn)生明顯的氧化性損傷。不同劑量的染毒組均能夠?qū)е赂闻K中活性氧(ROS)的升高,中、高劑量組肝臟中超氧化物歧化酶(SOD)、還原型谷胱甘肽(GSH)水平下調(diào)表明其抗氧化系統(tǒng)受到損傷,無法啟動(dòng)清除過量的ROS,但低劑量組小鼠肝臟啟動(dòng)其抗氧化機(jī)制,免于機(jī)體受到過量ROS的損傷。中、高劑量組小鼠肝臟中脂質(zhì)、蛋白、DNA均受到ROS的攻擊,造成氧化性損傷,機(jī)體處于氧化應(yīng)激狀態(tài),但低劑量AFB_1組小鼠體內(nèi)的生物大分子物質(zhì)并不受到顯著性的損傷。(3)AFB_1產(chǎn)生的氧化性損傷除導(dǎo)致細(xì)胞壞死外,凋亡也是一種可能的方式,且肝細(xì)胞凋亡率隨AFB_1濃度的增大而升高,在本試驗(yàn)范圍內(nèi)具有劑量-效應(yīng)關(guān)系。低劑量時(shí)AFB_1對(duì)細(xì)胞造成的影響主要以炎癥、局部細(xì)胞壞死為主,而較大劑量時(shí)則以細(xì)胞凋亡為主。AFB_1介導(dǎo)的肝細(xì)胞凋亡分子機(jī)制可能與上調(diào)Bax、Caspase-3、P53蛋白,下調(diào)Bcl-2蛋白的表達(dá)有關(guān)。AFB_1亞慢性染毒誘導(dǎo)的氧化性損傷中存在細(xì)胞凋亡的相關(guān)分子機(jī)制。這提示我們,若能從細(xì)胞凋亡的角度入手,更好的對(duì)其調(diào)控,將有可能為遏制AFB_1的氧化毒性提供新的途徑。(4)亞慢性條件下的AFB_1暴露能夠引起雄性小鼠肝細(xì)胞DNA鏈斷裂、DNA-DNA交聯(lián)、DNA-蛋白質(zhì)交聯(lián)形成,同時(shí)導(dǎo)致精子畸形率升高、微核形成及染色體損傷的增多。該五項(xiàng)指標(biāo)除DNA-DNA交聯(lián)外,其余各項(xiàng)指標(biāo)均隨AFB_1劑量的增大而升高,并且具有劑量-效應(yīng)關(guān)系,這說明AFB_1不僅對(duì)體細(xì)胞而且對(duì)生殖細(xì)胞也具有遺傳毒性,能產(chǎn)生致突變作用。(5)當(dāng)較大劑量的AFB_1暴露于小鼠時(shí)(1.5mg/kg),DNA-DNA交聯(lián)率并不隨AFB_1濃度的增大而升高。這一結(jié)果表明,當(dāng)AFB_1濃度增大到一定程度時(shí)(1.5mg/kg),其引發(fā)的各種DNA損傷形式中DNA-DNA交聯(lián)的比例下降,推測其他形式的DNA損傷(如DNA鏈斷裂、DNA-蛋白交聯(lián)等)所占比重增大。AFB_1產(chǎn)生遺傳毒性的機(jī)制可能是機(jī)體氧化應(yīng)激所產(chǎn)生大量ROS等自由基直接對(duì)DNA造成攻擊,或是ROS介導(dǎo)的氧化應(yīng)激間接導(dǎo)致DNA氧化損傷所形成的。(6)AFB_1不需經(jīng)過代謝,能夠直接與DNA之間發(fā)生相互作用,形成二元復(fù)合物。同時(shí)DNA能夠明顯淬滅AFB_1的內(nèi)源熒光,其淬滅類型為靜態(tài)結(jié)合動(dòng)態(tài)淬滅的方式。兩者的結(jié)合常數(shù)為4.12×103 L/mol;相互作用力主要為氫鍵和疏水相互作用;結(jié)合模式為溝槽結(jié)合。AFB_1的結(jié)合導(dǎo)致DNA的構(gòu)象有輕微的改變,但并沒有對(duì)其構(gòu)型造成較大的影響。分子對(duì)接結(jié)果說明,AFB_1與DNA的結(jié)合位于小溝槽區(qū),且AFB_1的O6和O3原子能夠和DA-17、DG16這兩個(gè)堿基形成三個(gè)較強(qiáng)的氫鍵。這提示我們,AFB_1對(duì)DNA造成的損傷可能存在新的作用方式,這種方式可能與其毒作用機(jī)制有關(guān)。綜上所述,亞慢性AFB_1的暴露能夠?qū)π∈蟮难趸⑦z傳系統(tǒng)造成一定程度的損傷,并且其作用機(jī)制都與機(jī)體處于氧化應(yīng)激狀態(tài)、大量ROS的生成有關(guān),這對(duì)于其致癌機(jī)制的研究與相關(guān)藥物的開發(fā)具有指導(dǎo)意義。
[Abstract]:Aflatoxin B_1 (Aflatoxin B_1, AFB_1) is the most toxic and most dangerous toxin of the known mycotoxins at present. Because of its strong carcinogenicity and its chemical structure stable at high temperature, it is difficult to break down, and AFB_1 exposure will eventually lead to the occurrence of acute or subchronic poisoning in various classes, although AFB_1 causes it. The role of cancer and its related mechanism has been confirmed, but the study on the oxidation, hereditary damage and related mechanism of the organism is not very clear. This is not conducive to the deep study of the toxic mechanism of Aflatoxins (AFT) and the establishment of the defense system of AFB_1. This experiment is on AFB_1 The oxidation, genetic toxicity and related molecular mechanisms were studied to provide a reference for the comprehensive assessment of the toxicity of AFB_1 and to elucidate the toxicity mechanism of other types of aflatoxin, and to provide more reference for the detoxification of aflatoxin or intoxication in vivo. 40 male mice were treated with 3 different doses of AFB_1 (0.09375,0.375,1.5mg/kg b.w./day) for 50 days under subchronic conditions. The oxidative damage and molecular mechanism caused by the oxidative damage and the related signaling pathway were evaluated for the oxidative damage and the related signaling pathways in the serum and the organs of the organs in order to evaluate the oxidative damage and the molecular mechanism. The DNA strand breaks in the liver. Cross-linking, cell micronucleus and sperm malformation were investigated to assess their genotoxicity and molecular mechanisms. The interaction mechanism between AFB_1 and DNA in vitro and the correlation with genetic toxicity were studied. The specific results are as follows: (1) after 50 days of AFB_1 exposure, AFB_1 can cause obvious organ damage to the mice The weight of ALT and AST increased significantly in the serum, and the weight of the three organs in the liver, kidney and testis also declined, indicating that the three organs were infected by AFB_1, and the pathological damage caused by.AFB_1 was low. In the high dose, the local necrosis of the hepatocytes, the swelling of the glomeruli and the necrosis of the renal tubular epithelial cells. (2) under subchronic conditions, AFB_1 can produce obvious oxidative damage to the liver of mice. The active oxygen (ROS) in the liver can be increased by the different doses of the infected group, and the high dose group of the liver is in the high dose group. The downregulation of superoxide dismutase (SOD) and reduced glutathione (GSH) showed that the antioxidant system was damaged and could not start to remove excess ROS, but the low dose group of mice liver started its antioxidant mechanism, so that the body was immune to excessive ROS. The lipid, protein, and DNA in the liver of the high dose group were attacked by ROS. In the oxidative stress, the body is in the oxidative stress state, but the biological macromolecules in the low dose AFB_1 mice are not significantly damaged. (3) the oxidative damage produced by AFB_1 is also a possible way of apoptosis in addition to cell necrosis, and the liver cell withering rate increases with the increase of AFB_1 concentration. There is a dose effect relationship within the circumference. The effects of AFB_1 on the cells at low doses are mainly inflammation and local cell necrosis, while the mechanism of apoptosis induced by apoptosis mainly mediated by.AFB_1 may be associated with the up regulation of Bax, Caspase-3, P53 protein, and the expression of Bcl-2 protein in.AFB_1 subchronic exposure There is a molecular mechanism of apoptosis in oxidative damage, which suggests that if we can regulate it from the angle of apoptosis, it will be possible to provide a new way to prevent the oxidative toxicity of AFB_1. (4) AFB_1 exposure in subchronic conditions can cause DNA strand breaks in male mice liver cells, DNA-DNA crosslinking, DNA- protein The formation of mass crosslinking resulted in the increase of sperm abnormality, the formation of micronucleus and the increase of chromosome damage. The five indexes, except for DNA-DNA cross-linking, were increased with the increase of the dose of AFB_1, and had a dose effect relationship, which indicated that AFB_1 not only had genetic toxicity to the somatic cells but also to the germ cells. Mutation. (5) when a large dose of AFB_1 was exposed to mice (1.5mg/kg), the cross-linking rate of DNA-DNA did not increase with the increase of AFB_1 concentration. This result showed that when the concentration of AFB_1 increased to a certain degree (1.5mg/kg), the proportion of DNA-DNA crosslinking in various forms of DNA damage caused by the AFB_1 decreased, and other forms of DNA damage (such as the DNA chain broken) were conjectured. The mechanism of increasing the proportion of.AFB_1 to increase the genetic toxicity of DNA- protein may be caused by a large number of free radicals such as ROS produced by the oxidative stress of the body, or the oxidative stress mediated by ROS, which indirectly leads to the oxidative damage of DNA. (6) AFB_1 does not need to be metabolizing and can interact directly with DNA. The two element composite is formed. At the same time, the internal fluorescence of AFB_1 can be quenched by DNA, and the quenching type is a static and dynamic quenching method. The binding constant of the two is 4.12 x 103 L/mol; the interaction force is mainly hydrogen bond and hydrophobic interaction, and the binding mode is a slight change in the conformation of DNA with the combination of groove and.AFB_1. The molecular docking results show that the combination of AFB_1 and DNA is located in the small groove area, and the O6 and O3 atoms of AFB_1 can form three strong hydrogen bonds with the two bases of DA-17 and DG16. This suggests that AFB_1 may have a new way of action on DNA caused by damage, which may be associated with its toxic agent. In summary, the exposure of subchronic AFB_1 can cause a certain degree of damage to the oxidation of mice and the genetic system, and its mechanism is related to the oxidative stress of the body and the production of a large number of ROS, which is of guiding significance for the research of its carcinogenic mechanism and the development of related drugs.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114

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