亚砷酸钠致大鼠胰岛β细胞DNA损伤机制的研究

发布时间：2018-03-28 18:55

本文选题：亚砷酸钠　切入点：大鼠胰岛细胞　出处：《大连医科大学》2012年硕士论文

【摘要】：前言：砷（As）是自然界中普遍存在的一种毒性很强的类金属元素。慢性砷中毒已成为全球面临的重大公共卫生问题，我国有很多慢性砷中毒病区。为了保障人群健康,世界卫生组织（WHO）、欧盟、日本和美国等先后将饮用水中砷的标准修订为10μg/L，我国新的《生活饮用水卫生标准》（2006年7月1日实施）也将砷含量标准由50μg/L降低到10μg/L。国际癌症研究机构（IARC）和美国疾病控制中心（CDC）已确定砷是第一类致癌物质。已有研究表明：无机砷可引起皮肤癌和肺癌，并且与肝癌、膀胱癌、肾脏癌、大肠癌的发生也密切相关。此外，有研究发现显示亚砷酸钠（NaAsO_2）具有遗传毒性，但有关遗传毒性机制的研究不多。研究表明，NaAsO_2可导致细胞中活性氧类（ROS）生成增多和8-羟基脱氧鸟苷（8-OHdG）表达升高。本研究通过观察不同浓度NaAsO_2对大鼠胰岛β细胞株INS-1内ROS和谷胱甘肽（GSH）水平、8-OHdG表达和DNA损伤的影响的影响，从氧化应激角度探讨砷所导致细胞DNA损伤的可能机制，为进一步的深入研究砷所引起的机体氧化损伤提供基础资料。方法：以INS-1细胞作为实验对象，通过单细胞凝胶电泳（SCGE）试验检测细胞DNA损伤情况，，评价NaAsO_2的遗传毒性：为探讨砷的遗传毒性机制，以2’,7’-二氢二氯荧光素（DCFH-DA）法和苯二醛（OPT）分别测定细胞内ROS以及GSH水平，以免疫组化方法检测细胞内8-OHdG的表达水平。实验结果用SPSS11.5统计。结果：NaAsO_2（2-16μmol/L）作用于INS-1细胞12h后，可引起DNA链断裂，细胞形成彗星样拖尾，拖尾细胞数明显增多，且呈剂量依赖关系。NaAsO_2（2-16μmol/L）作用于INS-1细胞12h后，可致8-OHdG的表达升高；NaAsO_2（2-16μmol/L）分别作用于INS-1细胞6h和12h后，可致细胞内ROS表达水平的显著增加，且呈剂量依赖关系，尤其是作用12h后，细胞内ROS表达水平最为显著；8μmol/L和16μmol/L NaAsO_2作用于INS-1细胞24h后，细胞内ROS表达水平增加；NaAsO_2（2-16μmol/L）分别作用于INS-1细胞48h后，细胞内ROS表达水平未见明显改变。NaAsO_2（2-16μmol/L）作用于INS-1细胞6，12，24和48h后，可致细胞内GSH表达水平的显著增加，且呈剂量依赖关系。结论：NaAsO_2通过氧化应激机制引起INS-1细胞DNA损伤。
[Abstract]:The arsenic (As) is a kind of toxic metal elements exist in nature is very strong. Chronic arsenic poisoning has become a major public health problem facing the world, our country has a lot of chronic arsenic poisoning area. In order to protect people's health, the WHO (WHO), the European Union, Japan and the United States has to be revised in drinking water the arsenic standard is 10 g/L, the < > new standard for drinking water quality in China (July 1, 2006) will also be the standard for arsenic decreased from 50 g/L to 10 g/L.
The international agency for research on cancer (IARC) and the US Centers for Disease Control (CDC) has identified arsenic is the first class of carcinogens. Previous studies have shown that arsenic can cause skin cancer and lung cancer, and liver cancer, bladder cancer, kidney cancer, is also closely related to the incidence of colorectal cancer. In addition, studies have found that sodium arsenite (display NaAsO_2) with genetic toxicity, but the research on the mechanism of genotoxicity is not much. The results show that NaAsO_2 can lead to reactive oxygen species (ROS) generation cells increased and 8- hydroxydeoxyguanosine (8-OHdG) expression.
This study through the observation of different concentrations of NaAsO_2 on rat islet beta cell line INS-1 ROS and glutathione (GSH) level, influence the expression of 8-OHdG and DNA damage, arsenic caused injury of DNA cells from oxidative stress, to provide the basic data for the oxidative damage caused by arsenic and further research on the.
Methods: INS-1 cells were used as experimental objects, by single cell gel electrophoresis (SCGE) assay for detection of cell DNA damage and genetic toxicity evaluation NaAsO_2: genetic toxicity in order to explore the mechanism of arsenic, 2 ', 7' - two hydrogen chloride two fluorescein (DCFH-DA) method and two benzene aldehyde (OPT) in ROS cells and GSH levels were measured, the expression level was detected by immunohistochemistry in cell 8-OHdG. The experimental results with SPSS11.5 statistics.
Results: NaAsO_2 (2-16 mol/L) in INS-1 cells after 12h induced DNA strand breaks, cells form a comet like tail, tail and decreased the number of cells in a dose-dependent manner.NaAsO_2 (2-16 mol/L) in INS-1 cells after 12h may induce the expression of 8-OHdG increased; NaAsO_2 (2-16 mol/L) respectively. The role of 6H and 12h in INS-1 cells, may induce the expression of intracellular ROS levels increased significantly, in a dose-dependent manner, especially after 12h cells, the expression level of ROS was most significant; 8 mol/L and 16 mol/L NaAsO_2 in INS-1 cells after 24h, the expression of the intracellular ROS level increased (NaAsO_2; 2-16 mol/L) respectively in INS-1 cells after 48h, the expression level of.NaAsO_2 had no obvious change in ROS cells (2-16 mol/L) in INS-1 cells of 6,12,24 and 48h, may induce the expression of intracellular GSH levels increased significantly, in a dose-dependent manner.
Conclusion: NaAsO_2 induced DNA damage in INS-1 cells through oxidative stress mechanism.

【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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