棒曲霉素引起HEK-293细胞DNA损伤的溶酶体途径

发布时间：2018-05-05 01:20

本文选题：棒曲霉素 + DNA链断裂　；参考：《大连医科大学》2012年硕士论文

【摘要】：目的：棒曲霉素（Patulin,PAT），又称为展青霉素，属于真菌毒素类，主要是曲霉属、青霉属、裸囊菌属等真菌的次生代谢产物。棒曲霉素在1940年第一次被分离出来，经过多年的研究，在近些年人们逐渐认识到这种棒曲霉素是存在于苹果和苹果产品中的毒素，并成为全球广泛关注的霉素。国际癌症研究中心（International Agency for Research on Cancer,IARC）将PAT对人类的致癌性列为第三类。世界卫生组织(WHO)规定了食品中棒曲霉素的最高限量为50μg/L,而目前欧盟已有更严格要求的趋势，50μg/kg为棒曲霉素残留限定量，对婴幼儿食品内含量限定为10μg/kg体重。棒曲霉素经过毒理学试验证明其能使动物畸变、突变和癌变。研究还表明PAT还有遗传毒性和免疫毒性。有研究显示PAT的遗传毒性与细胞的氧化应激有关。近期研究证明了DNA的损伤与溶酶体膜稳定性改变也有关系。因此，我们研究了棒曲霉素引起的DNA损伤与细胞内ROS水平以及溶酶体的关系，，旨在探讨PAT遗传毒性的机制。本研究选用人胚肾HEK-293细胞，探讨棒曲霉素的DNA损害作用可能机制，为进一步评估PAT对人类的健康危害提供实验资料。方法：以HEK-293细胞作为试验系统。通过MTT试验检测PAT对HEK-293细胞毒性大小，通过单细胞凝胶电泳（SCGE）试验检测细胞DNA损伤情况，评价PAT遗传毒性。为探讨其可能的遗传毒性机制，用DCFH法检测细胞内ROS水平，用吖啶橙（Acridine orange）测定细胞内溶酶体膜稳定性；采用N-乙酰半胱氨酸（NAC）和氯化铵（NH4CL）对溶酶体进行保护干预；分别采用NAC、NH4CL、抑胃肽（pepstatin A）干预PAT所致的DNA损伤。实验结果用SPSSv11.5统计软件进行统计分析。结果：5-20μM的PAT作用于HEK-293细胞1h后，引起细胞DNA链断裂，细胞形成彗星样拖尾，其尾长、尾矩和尾百分含量与PAT呈剂量依赖关系；2.5-40μM的PAT作用于HEK-293细胞1h后引起细胞内溶酶体膜稳定性发生改变，80μM的PAT作用1h后引起ROS水平增加。用10mM的NH4CL、500mM的NAC预处理HEK-293细胞1h后，都明显保护了细胞内溶酶体膜稳定性。分别用10mM的NH4CL、500mM的NAC和100μM的pepstatin A预处理HEK-293细胞1h后，PAT引起的DNA链断裂几乎完全被阻止。但用30μM的地昔帕明预处理后，PAT引起的DNA链断裂没有得到明显改善，可能与单独地昔帕明引起了HKE-293细胞DNA损伤有关。结论：棒曲霉素可致HEK-293细胞DNA链断裂，其作用机制可能与溶酶体途径有关，通过溶酶体膜稳定性的破坏释放一些溶酶体水解酶，从而导致DNA链断裂。NAC是有效的抗氧化剂，能与NH4CL同时保护溶酶体膜的稳定性，说明可能ROS的产生是PAT本身的毒性导致的，并通过溶酶体途径引起DNA链断裂。溶酶体可能是PAT细胞毒性的生物靶点之一，并由ROS引起膜稳定性的破坏。
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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