本文選題：棒曲霉素 + DNA鏈斷裂��； 參考：《大連醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：棒曲霉素（Patulin,PAT），又稱為展青霉素，屬于真菌毒素類，主要是曲霉屬、青霉屬、裸囊菌屬等真菌的次生代謝產(chǎn)物。棒曲霉素在1940年第一次被分離出來，經(jīng)過多年的研究，在近些年人們逐漸認(rèn)識(shí)到這種棒曲霉素是存在于蘋果和蘋果產(chǎn)品中的毒素，并成為全球廣泛關(guān)注的霉素。 國際癌癥研究中心（International Agency for Research on Cancer,IARC）將PAT對人類的致癌性列為第三類。世界衛(wèi)生組織(WHO)規(guī)定了食品中棒曲霉素的最高限量為50μg/L,而目前歐盟已有更嚴(yán)格要求的趨勢，50μg/kg為棒曲霉素殘留限定量，對嬰幼兒食品內(nèi)含量限定為10μg/kg體重。 棒曲霉素經(jīng)過毒理學(xué)試驗(yàn)證明其能使動(dòng)物畸變、突變和癌變。研究還表明PAT還有遺傳毒性和免疫毒性。有研究顯示PAT的遺傳毒性與細(xì)胞的氧化應(yīng)激有關(guān)。近期研究證明了DNA的損傷與溶酶體膜穩(wěn)定性改變也有關(guān)系。因此，我們研究了棒曲霉素引起的DNA損傷與細(xì)胞內(nèi)ROS水平以及溶酶體的關(guān)系，，旨在探討PAT遺傳毒性的機(jī)制。 本研究選用人胚腎HEK-293細(xì)胞，探討棒曲霉素的DNA損害作用可能機(jī)制，為進(jìn)一步評估PAT對人類的健康危害提供實(shí)驗(yàn)資料。 方法：以HEK-293細(xì)胞作為試驗(yàn)系統(tǒng)。通過MTT試驗(yàn)檢測PAT對HEK-293細(xì)胞毒性大小，通過單細(xì)胞凝膠電泳（SCGE）試驗(yàn)檢測細(xì)胞DNA損傷情況，評價(jià)PAT遺傳毒性。為探討其可能的遺傳毒性機(jī)制，用DCFH法檢測細(xì)胞內(nèi)ROS水平，用吖啶橙（Acridine orange）測定細(xì)胞內(nèi)溶酶體膜穩(wěn)定性；采用N-乙酰半胱氨酸（NAC）和氯化銨（NH4CL）對溶酶體進(jìn)行保護(hù)干預(yù)；分別采用NAC、NH4CL、抑胃肽（pepstatin A）干預(yù)PAT所致的DNA損傷。實(shí)驗(yàn)結(jié)果用SPSSv11.5統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析。 結(jié)果：5-20μM的PAT作用于HEK-293細(xì)胞1h后，引起細(xì)胞DNA鏈斷裂，細(xì)胞形成彗星樣拖尾，其尾長、尾矩和尾百分含量與PAT呈劑量依賴關(guān)系；2.5-40μM的PAT作用于HEK-293細(xì)胞1h后引起細(xì)胞內(nèi)溶酶體膜穩(wěn)定性發(fā)生改變，80μM的PAT作用1h后引起ROS水平增加。用10mM的NH4CL、500mM的NAC預(yù)處理HEK-293細(xì)胞1h后，都明顯保護(hù)了細(xì)胞內(nèi)溶酶體膜穩(wěn)定性。分別用10mM的NH4CL、500mM的NAC和100μM的pepstatin A預(yù)處理HEK-293細(xì)胞1h后，PAT引起的DNA鏈斷裂幾乎完全被阻止。但用30μM的地昔帕明預(yù)處理后，PAT引起的DNA鏈斷裂沒有得到明顯改善，可能與單獨(dú)地昔帕明引起了HKE-293細(xì)胞DNA損傷有關(guān)。 結(jié)論：棒曲霉素可致HEK-293細(xì)胞DNA鏈斷裂，其作用機(jī)制可能與溶酶體途徑有關(guān)，通過溶酶體膜穩(wěn)定性的破壞釋放一些溶酶體水解酶，從而導(dǎo)致DNA鏈斷裂。NAC是有效的抗氧化劑，能與NH4CL同時(shí)保護(hù)溶酶體膜的穩(wěn)定性，說明可能ROS的產(chǎn)生是PAT本身的毒性導(dǎo)致的，并通過溶酶體途徑引起DNA鏈斷裂。溶酶體可能是PAT細(xì)胞毒性的生物靶點(diǎn)之一，并由ROS引起膜穩(wěn)定性的破壞。
[Abstract]:Objective: patulinella patulinensis, also called aspericillins, belongs to mycotoxins, mainly secondary metabolites of Aspergillus, Penicillium and Phaeocystis. Patulin was isolated for the first time in 1940. After many years of research, it has been gradually recognized that patulin is a toxin in apple and apple products, and has become a worldwide concern. The International Agency for Research on Cancer (IARC) lists PAT's carcinogenicity in humans as category III. The World Health Organization (WHO) stipulates that the maximum limit of patulin in food is 50 渭 g / L, while the EU has a more stringent trend that 50 渭 g/kg is the limit of patulin residue, and the content of baby food is limited to 10 渭 g/kg body weight. Patulin has been proved to be capable of distorting, mutating and cancerizing animals by toxicological tests. Studies have also shown that PAT also has genotoxicity and immune toxicity. Studies have shown that the genotoxicity of PAT is associated with oxidative stress in cells. Recent studies have demonstrated that DNA damage is also associated with changes in the stability of lysosomal membranes. Therefore, we studied the relationship between DNA damage induced by patulin and intracellular ROS levels and lysosomes in order to explore the mechanism of PAT genotoxicity. In this study, human embryonic kidney HEK-293 cells were selected to investigate the possible mechanism of DNA damage of patulin, and to provide experimental data for further evaluation of PAT harm to human health. Methods: HEK-293 cells were used as test system. The cytotoxicity of PAT to HEK-293 cells was detected by MTT test, and the DNA damage was detected by single cell gel electrophoresis (SCGE) test to evaluate the genetic toxicity of PAT. In order to study the possible genotoxic mechanism, the intracellular ROS level was detected by DCFH assay, the stability of lysosomal membrane was determined by acridine orange (Acridine orange), and the lysosomal protection was carried out by N-acetylcysteine (NAC) and ammonium chloride (NH4CLL). The DNA injury induced by PAT was treated with NACN NH4 CLA and pepstatin A, respectively. The experimental results were analyzed by SPSSv11.5 software. Results after treated with PAT of 5 ~ 20 渭 M for 1 h, the DNA strand of HEK-293 cells was broken, and the cells formed comet-like tail, and the tail was long. The caudal moment and the percentage content of the tail were in a dose-dependent relationship with PAT. After treated with 2.5-40 渭 M PAT for 1 h, the stability of lysosomal membrane of HEK-293 cells was changed. The ROS level was increased after the treatment of 80 渭 M PAT for 1 h. Pretreatment of HEK-293 cells with 10mM NH _ 4CLN 500mm NAC for 1 h significantly protected the stability of intracellular lysosomal membrane. The DNA strand breaks induced by pat were almost completely prevented by pretreatment of HEK-293 cells with 10mM NH _ 4CLN 500mm NAC and 100 渭 M pepstatin A for 1 h, respectively. However, the DNA strand breaks induced by pat were not significantly improved after preconditioning with 30 渭 M of dioxipramine, which may be related to the DNA damage of HKE-293 cells induced by dixipramine alone. Conclusion: patulin can induce DNA strand break in HEK-293 cells, and its mechanism may be related to lysosomal pathway. Lysosomal hydrolase is released by destroying the stability of lysosomal membrane, resulting in DNA strand break. NAC is an effective antioxidant. It can protect the stability of lysosomal membrane at the same time as NH4CL, which indicates that the production of ROS may be caused by the toxicity of PAT itself, and DNA strand break is induced by lysosome pathway. Lysosomes may be one of the biological targets of PAT cytotoxicity, and the stability of membrane may be destroyed by ROS.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R114

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