喹乙醇诱导胰岛细胞DNA损伤及其机制探讨

发布时间：2018-01-15 13:02

本文关键词：喹乙醇诱导胰岛细胞DNA损伤及其机制探讨　出处：《大连医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的：喹乙醇(olaquindox),是一种抗菌促生长剂，被广泛应用在禽畜和水产养殖业，，已有实验表明喹乙醇对肝肾均有损害，但尚未有关于喹乙醇对胰岛损害的研究，本实验通过彗星实验观察喹乙醇是否会对大鼠胰岛细胞的DNA有损伤并探讨其损害可能的机制，通过western blot实验观察喹乙醇对p53蛋白表达的影响，从而探讨由持续DNA损伤可能引起的与癌有关基因表达的增加，为更全面的评估喹乙醇多脏器的损害提供依据。方法：喹乙醇引起的DNA损伤是通过单细胞凝胶电泳实验检测，用2，7，-二氢二氯荧光素（DCFH）检测细胞内活性氧（ROS）含量，用吖啶橙（AO）检测细胞内溶酶体膜稳定性，通过辣根过氧化物酶标记的荧光指示剂来检测westernblot实验中P53蛋白表达量，通过碱性保护剂NH4Cl和NAC来干预彗星实验及分别干预溶酶体实验和ROS实验。结果：喹乙醇（0~1600ug/ml）作用于INS-1细胞1h后，彗星实验中尾长、尾距、尾DNA％含量均增加，并呈剂量依赖，在对800、1600ug/ml剂量组分别加NH4Cl和NAC干预后均有保护作用。喹乙醇同样作用于INS-1细胞1h，可使细胞内溶酶体膜稳定性下降、ROS含量增加，分别用NH4Cl和NAC干预后均有保护作用。喹乙醇分别作用于INS-1细胞24h和48h后，P53蛋白表达量增加。结论：喹乙醇可以诱导大鼠胰岛细胞DNA损伤，对大鼠胰岛细胞有遗传毒性， NAC和NH4Cl均可对喹乙醇所致的DNA损伤起到保护作用，说明喹乙醇DNA损伤的途径是氧化应激和溶酶体途径。喹乙醇可使p53蛋白表达量增加，表明喹乙醇可使与癌有关基因表达增加，其机制可能是DNA的持续损伤。
[Abstract]:Objective: olaquindox (olaquindox), is a kind of antibacterial auxogen, is widely used in livestock and aquaculture, previous studies have demonstrated that the damage to the liver and kidney of olaquindox, but not yet a study of olaquindox on islet lesions, the experimental observation of the comet will ethanol on rat islet cell damage and DNA to explore the possible mechanism of the damage effect of Western blot through the experimental observation of olaquindox on expression of p53 protein increased, so as to explore the possible damage caused by sustained DNA cancer related gene expression, and provide the basis for a more comprehensive assessment of olaquindox multiple organ damage.
Methods: DNA damage caused by olaquindox by single cell gel electrophoresis assay, 2,7, two - two hydrogen chloride fluorescein (DCFH) detection of intracellular reactive oxygen species (ROS) content, using acridine orange (AO) to detect the intracellular lysosomal membrane stability by horseradish peroxidase labeled fluorescent indicator to detect the expression of P53 in mind the amount of protein in the Westernblot experiment, the protective agent NH4Cl and NAC by alkaline comet assay and intervention intervention respectively lysosomes and ROS experiment.
Results: olaquindox (0~1600ug/ml) in INS-1 cells after 1h, the comet tail length, tail length, the tail DNA% content were increased in a dose-dependent manner, the protective effect of 8001600ug/ml was NH4Cl and NAC respectively with the dose group after the intervention. The same effect on INS-1 cells of olaquindox 1H, the lysosomal membrane stability decreased, ROS content increased by NH4Cl and NAC, respectively. After the intervention has a significant protective effect of olaquindox were acting in 24h and 48h INS-1 cells, the expression of P53 protein was increased.
Conclusion: olaquindox can islet cell injury in rats induced by DNA, genetic toxicity on rat islet cells, NAC and NH4Cl on DNA damage induced by olaquindox can play a protective role, how olaquindox DNA injury is oxidative stress and lysosomal pathway. Olaquindox can make the expression of p53 protein was increased, and that of olaquindox can make cancer the increased gene expression, its mechanism may be sustained damage in DNA.

【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

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