两种不同砷化物对大鼠胰岛β细胞株凋亡机制的探索

发布时间：2018-03-08 21:26

本文选题：砷化物　切入点：大鼠胰岛β细胞　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：砷是普遍存在于自然界中毒性很强的类金属元素，人类主要从饮水、食物和土壤中摄取砷，慢性砷中毒已成为全球面临的重大公共卫生问题。近年有流行病学研究显示砷与糖尿病之间存在关联，研究显示砷导致糖尿病的主要机制可能是破坏胰岛β细胞功能导致细胞损伤或凋亡。砷化物的化学形式分为无机砷和有机胂，目前研究表明，，砷化物的毒性高度依赖于它的化学形式，不同形态的砷在细胞内代谢途径不同，具有不同的毒理学性质。尽管砷暴露作为糖尿病的危险因素已被认可，但迄今为止其生物效应机制仍未完全研究清楚，且实验室数据多为研究三价无机砷与糖尿病之间的关联。为了进一步的探索砷化物和糖尿病之间的关系和可能机制，本研究试图观察不同砷化物对体外培养大鼠胰岛β细胞株（INS-1）的毒性作用，为砷导致糖尿病的发病机制研究提供线索。方法：本实验选定大鼠胰岛β细胞株（INS-1），通过MTT法检测砷化物对胰岛细胞的毒性作用。用annexin V-FITC/PI流式细胞仪检测法和Hoechst33258细胞染色法检测不同砷化物致细胞凋亡情况。用2’,7’-二氢二氯荧光素（DCFH）检测细胞内活性氧(ROS)的含量，用Western blot免疫印迹蛋白法检测细胞内P53的蛋白含量变化。实验结果用SPSS13.0统计软件包进行统计分析。结果：高剂量暴露五价无机砷、五价二甲基胂（分别50μmol/L、100μmol/L）能够降低INS-1的细胞生存率，且iAs~(5-)的细胞毒性强于DMA~(5+)；iAs~(5+)（50-200μmol/L）和DMA~(5+)（100-400μmol/L）作用于INS-1细胞48h后，最高剂量暴露组均有凋亡细胞明显的增多现象，这一实验结果提示高浓度的iAs~(5+)和DMA~(5+)暴露使细胞发生凋亡；高剂量iAs~(5+)、DMA~(5+)暴露24h均可以诱发INS-1细胞内ROS水平的显著升高，并且药物的剂量存在依赖关系，提示在iAs~(5+)、DMA~(5+)作用于细胞的初期，细胞内发生了氧化应激反应；检测发现经iAs~(5+)染毒的INS-1细胞核内p53蛋白表达增多，而经DMA~(5+)染毒的INS-1细胞核内p53蛋白表达无明显变化。结论：不同砷化物（砷酸氢二钠、二甲基胂酸钠）对大鼠胰岛β细胞的毒性作用及机制不同。
[Abstract]:Objective: arsenic is a highly toxic metalloid element in nature. People mainly take arsenic from drinking water, food and soil. Chronic arsenism has become a major public health problem worldwide. Epidemiological studies in recent years have shown a link between arsenic and diabetes. Studies have shown that the main mechanism of arsenic induced diabetes may be the destruction of islet 尾 cell function leading to cell damage or apoptosis. The chemical forms of arsenide are inorganic arsenic and organic arsine. The toxicity of arsenide is highly dependent on its chemical form. Different forms of arsenic have different metabolic pathways in cells and have different toxicological properties, although arsenic exposure has been recognized as a risk factor for diabetes. However, the mechanism of its biological effect has not been fully studied, and most of the laboratory data are related to the relationship between trivalent inorganic arsenic and diabetes mellitus, in order to further explore the relationship and possible mechanism between arsenide and diabetes, This study attempts to observe the toxic effects of different arsenides on rat islet 尾 cell line INS-1 in vitro, which provides clues for the study of the pathogenesis of arsenic induced diabetes. Methods: rat islet 尾 cell line INS-1 was selected to detect the toxicity of arsenide to islet cells by MTT method. Apoptosis induced by different arsenic compounds was detected by annexin V-FITC / Pi flow cytometry and Hoechst33258 cell staining. The content of reactive oxygen species (Ros) in the cells was determined by DCFH- DCFH- Dichlorofluorescein dihydrodichlorofluorescein (DCFH). The changes of p53 protein in cells were detected by Western blot Western blot method. The results were analyzed by SPSS13.0 software package. Results: high dose exposure to pentavalent inorganic arsenic, pentavalent dimethyl arsine (50 渭 mol / L, 100 渭 mol / L, respectively) decreased the survival rate of INS-1 cells, and the cytotoxicity of iAsN 5 was stronger than that of DMA~(5 (50 ~ 200 渭 mol / L) and DMA~(5 (100 ~ 400 渭 mol / L) for 48 h. The results showed that high concentration of iAs~(5) and DMA~(5) could induce apoptosis of INS-1 cells, and high dose of iAs~(5 could induce the increase of ROS level in INS-1 cells after 24 hours of exposure. Moreover, the dose-dependence of the drug indicated that the oxidative stress reaction occurred in the cells at the early stage of the action of iAs~(5, and the expression of p53 protein in the nucleus of INS-1 exposed to iAs~(5) was increased. However, the expression of p53 protein in the nucleus of INS-1 exposed to DMA~(5 had no significant change. Conclusion: the toxicity and mechanism of different arsenides (disodium arsenate, dimethylarsonic acid) on islet 尾 cells of rats are different.
【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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