硫酸铟对L929细胞毒性机制的研究

发布时间：2018-06-15 03:11

本文选题：硫酸铟 + DNA损伤　；参考：《苏州大学》2012年硕士论文

【摘要】：目的：铟（indium,In）是一种稀散金属，在信息宇航能源军事工业及医药卫生领域均有着很广泛的应用。随着铟及其化合物的使用增多，，接触人群也在不断扩大。通过各种途径接触吸收的铟通过血液转运到软组织及骨骼，并随尿及粪便排出体外。本研究旨在了解硫酸铟的细胞毒性DNA损伤及细胞毒性机制，试图探讨硫酸铟可能的细胞损伤机制，为提出有效保护职业暴露人群健康的措施提供基础依据。方法：(1)以小鼠成纤维细胞（L929）作为实验系统，采用四甲基偶氮唑盐法（MTT比色法）检测不同浓度硫酸铟对L929细胞作用不同时间段后，细胞存活率的变化，了解浓度-时间-毒性效应的关系；（2）采用单细胞凝胶电泳实验（SCGE彗星实验）研究不同浓度硫酸铟对L929细胞DNA单链断裂损伤的程度；（3）采用免疫荧光技术研究不同浓度硫酸铟对L929细胞DNA双链断裂损伤的程度；（4）用流式细胞技术DCFH-DA法检测不同浓度硫酸铟处理L929细胞后活性氧（ROS）含量的变化；（5）流式细胞术用JC-1检测不同浓度硫酸铟作用下对L929细胞线粒体膜电位的改变。结果：（1）MTT法显示，在24h及48h处理过程中，当硫酸铟终浓度1mmol/L及以上，各染毒组吸光度值与阴性对照组相比较具有统计学差异（P 0.05）。随染毒浓度增加，细胞存活率也呈浓度依赖性降低。（2）单细胞凝胶电泳结果显示，在1～4mmol/L硫酸铟作用2h及12h后，彗星细胞拖尾率随着浓度的增加而增加，且有统计学意义（P 0.05）。其他分析指标彗星尾长、Olive尾矩和彗尾DAN含量也明显高于阴性对照组(P 0.05)。（3）免疫荧光实验显示：在1～6mmol/L硫酸铟作用2h后，细胞核内γH2AX荧光强度明显增强，焦点数增加，与阴性对照组比较差异具有统计学意义（P 0.05）。（4）流式细胞仪检测结果显示，随硫酸铟染毒浓度升高，活性氧（ROS）含量相应升高并呈现线性相关关系。（5）经流式细胞仪检测，在硫酸铟染毒12h条件下，随染毒浓度增加线粒体膜电位水平降低，且差异具有统计学意义(P 0.05)。结论：（1）在体外培养的条件下，硫酸铟能明显抑制L929细胞的增殖。在各个染毒时间段内细胞存活率随染毒浓度增加而降低，并呈浓度依赖关系。（2）1mmol/L硫酸铟染毒2h可引起细胞DNA单链断裂。（3）在2h染毒条件下硫酸铟可引起细胞DNA双链断裂。（4）硫酸铟可引起L929细胞内活性氧含量增加。（5）硫酸铟可引起L929细胞线粒体膜电位降低。本次研究结果显示，硫酸铟可引起体外培养的L929细胞活性氧含量增加，造成DNA损伤，同时引起膜电位降低，影响线粒体稳定性。
[Abstract]:Objective: indium Inis is a dilute metal in information? Space? energy Military industry and medical and health fields have a very wide range of applications. As the use of indium and its compounds increases, the number of people exposed to it is expanding. Indium absorbed through various ways is transported through blood to soft tissues and bones and discharged with urine and faeces. The purpose of this study was to understand the cytotoxic DNA damage and cytotoxic mechanism of indium sulfate, and to explore the possible mechanism of cell damage in indium sulfate, and to provide the basis for effective measures to protect the health of occupational exposed population. Methods Mouse fibroblasts (L929) were used as experimental system. MTT colorimetric assay was used to detect the survival rate of L929 cells treated with different concentrations of indium sulfate for different time periods. To understand the relationship between concentration, time and toxicity, the single cell gel electrophoresis (SCGE) assay and comet assay were used to study the damage degree of DNA single strand break in L929 cells with different concentrations of indium sulfate. (3) Immunofluorescence technique was used to study the degree of DNA double strand break damage in L929 cells treated with different concentrations of indium sulfate. Flow cytometry (DCFH-DA) was used to detect the changes of reactive oxygen species (Ros) content in L929 cells treated with different concentrations of indium sulfate. Flow cytometry was used to detect the mitochondrial membrane potential of L929 cells treated with different concentrations of indium sulfate. Results in 24 h and 48 h treatment, when the final concentration of indium sulfate was 1 mmol / L or above, the absorbance value of each group was significantly different from that of negative control group (P 0.05). The survival rate of comet cells decreased in a concentration-dependent manner with the increase of concentration. The results of single cell gel electrophoresis showed that the trailing rate of comet cells increased with the increase of concentration after 2 h and 12 h exposure to 1 mol / L indium sulfate, and there was statistical significance (P 0.05). Olive tail moment and DAN content in comet tail were also significantly higher than those in negative control group (P 0.05). The results of immunofluorescence assay showed that the fluorescence intensity of 纬 H 2AX and the number of focal points in the nucleus were significantly increased after 2 h exposure to 1 渭 mol / L indium sulfate. The results of flow cytometry showed that the content of reactive oxygen species rose with the increase of indium sulfate concentration and showed a linear correlation with flow cytometry. When treated with indium sulfate for 12 h, the level of mitochondrial membrane potential decreased with the increase of concentration of indium sulfate, and the difference was statistically significant (P 0.05). Conclusion in vitro culture, indium sulfate can significantly inhibit the proliferation of L929 cells. The cell survival rate decreased with the increase of the exposure concentration during each exposure period. In a concentration-dependent manner, 1 mmol / L indium sulfate could induce single strand breaks of DNA in L929 cells for 2 h.) under the condition of 2 h exposure, indium sulfate could induce double strand breaks of DNA. 4) indium sulfate could increase the content of reactive oxygen species in L929 cells. Mitochondrial membrane potential of L 929 cells decreased. The results showed that indium sulfate could increase the content of reactive oxygen species in cultured L929 cells, cause DNA damage and decrease the membrane potential and affect the stability of mitochondria.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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