胶霉毒素对HEK-293细胞氧化应激性DNA损伤作用及机制探讨

发布时间：2018-07-23 10:49

【摘要】：目的：胶霉毒素（GT）广泛存在于里氏木霉、青霉、烟曲霉和白色念珠菌等致病菌或环境微生物的次生代谢产物中，研究证明GT的产生主要来源于烟曲霉毒素，，另外通过液相色谱串联质谱（HPLC）的方法检测发现胶霉毒素存在于动物（如猪，奶牛，家禽）的饲料中，也产生于玉米的贮存过程。体外实验表明胶霉毒素有很强的抗菌、抗病毒作用,呈现出较好的药用前景，曾一时被认为可用于抗肿瘤治疗。但随后的研究表明胶霉毒素具有抑制血管生成、免疫抑制，抑制NF-kB及其他特定基因表达及介导细胞凋亡等毒性。本研究通过观察胶霉毒素诱导HEK-293细胞的遗传毒性，以及其引起ROS的产生和溶酶体膜通透的改变，旨在探讨GT对DNA损伤的可能毒性机制，为进一步评估胶霉毒素对人类的健康影响提供试验及理论依据。方法：以HEK-293细胞作为试验系统。本实验运用单细胞凝胶电泳试验（SCGE）和蛋白质印迹法（western blot）检测GT对体外HEK-293细胞DNA损伤情况以及DNA损伤蛋白的高表达（ATM和P53)水平，进而验证胶霉毒素的DNA损伤毒性。为了进一步探讨其可能的DNA损伤机制，以2’,7’—二氢二氯荧光素（DCFH）和吖啶橙（AO）分别测定胶霉毒素对细胞内活性氧（ROS）以及溶酶体膜稳定性的影响，通过N-乙酰半胱氨酸（NAC）干预试验观察氧化性损伤在DNA损伤中的作用，以及对溶酶体膜稳定性的影响。试验结果用SPSS v12.7统计软件进行统计分析。结果：当胶霉毒素（GT)作用于HEK-293细胞1小时后，彗星试验显示其引起DNA双链断裂，荧光显微镜下与DMSO空白对照组相比，细胞成彗星样拖尾，其趋势随着浓度而递增，其中尾长增长，尾DNA%以及DNA%含量明显增大；同时，GT（0.3125μM～1.25μM）可导致细胞内ROS的表达水平升高，溶酶体膜稳定性下降。用NAC干预剂预处理后，GT所引起的HEK293细胞的拖尾现象、细胞内ROS以及溶酶体膜通透都有明显地减弱。作用24小时后，胶霉毒素同样能诱导HEK293细胞的DNA损伤，其损伤蛋白的表达水平呈剂量依赖效应趋势，而此时未出现HEK293细胞的凋亡发生，DNA损伤蛋白ATM/P53的表达水平可被抗氧化剂NAC有效干预，结果出现DNA损伤高表达蛋白明显的下降趋势。结论：胶霉毒素在高浓度0.625μM-1.25μM的作用下能够引起HEK293细胞的DNA损伤，说明其具有遗传毒性。同时，GT可导致HEK-293细胞内ROS生成增加，使细胞处于氧化应激状态，并使溶酶体膜稳定性下降。而抗氧化剂NAC在降低了细胞内ROS水平的同时，对溶酶体膜稳定性呈现保护趋势，同样条件下减弱了GT诱导的DNA链断裂程度，这些结果表明胶霉毒素能引起HEK-293细胞内溶酶体膜稳定性下降及DNA损伤，其作用机制与氧化应激性有关。DNA损伤蛋白（ATM，P53）的高表达以及NAC对其的保护作用，进一步说明了胶霉毒素能引起氧化性DNA损伤。
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

【共引文献】