二甲基甲酰胺对V79细胞的毒性作用研究

发布时间：2018-04-18 04:11

本文选题：二甲基甲酰胺 + MTT　；参考：《苏州大学》2013年硕士论文

【摘要】：目的：二甲基甲酰胺(N, N-dimethylformamide, DMF)是一种低毒类的有机溶剂，是一种重要的化工原料，目前广泛应用于有机合成、石油提炼等工业行业，以及医药和其他行业。随着现代工业的发展，DMF的应用范围不断扩大，职业接触人员也不断增加，这给接触DMF的作业人群带来了健康安全隐患。本研究通过了解二甲基甲酰胺（DMF）对中国仓鼠肺细胞（V79细胞）的细胞毒性、DNA损伤作用以及细胞毒性机制，试图探讨二甲基甲酰胺可能的细胞损伤机制，为提出有效的保护职业暴露人群健康的措施提供依据。方法：（1）以体外培养V79细胞为研究对象，采用四氮唑盐比色分析法（MTT法）检测不同浓度的DMF对V79细胞作用不同时间段后，细胞存活率的变化，了解浓度-时间-毒性效应关系；（2）采用碘化吡啶（PI)染色观察细胞周期分布状况，了解DMF对V79细胞周期的影响；（3）用碱性单细胞凝胶电泳技术（SCGE）检测不同浓度DMF在作用不同时间段后，V79细胞的DNA单链断裂的损伤情况。（4）用中性单细胞凝胶电泳技术检测不同浓度DMF在作用不同时间段后，V79细胞的DNA双链断裂的情况；(5)用流式细胞术DCFH-DA法检测不同浓度DMF处理V79细胞后活性氧（ROS）含量比的变化。结果：(1) MTT结果显示，各组V79细胞存活率随染毒浓度及染毒时间的增加而下降，与阴性组比较差异均有统计学意义（P＜0.05），存在明显的剂量-效应关系（6h, β=-0.002，P＜0.05;12h, β=-0.003，P＜0.05;24h, β=-0.003，P＜0.05）。(2)流式细胞仪检测细胞周期发现，经DMF作用24h后V79细胞周期发生了明显的变化。每一染毒时间段内，，随着DMF染毒浓度的增加，G1期细胞的构成比降低；而S期细胞和G2期细胞的比例则随着DMF浓度的升高而升高，各染毒组与阴性对照组比较均有差异性，差异具有统计学意义（P＜0.05）。(3)碱性单细胞凝胶电泳结果显示，彗星拖尾率、尾长、Olive尾距、尾部DNA百分含量随着DMF染毒浓度的增加而增大，经SNK方差分析，各染毒浓度组和同组阴性组比较，差异均有统计学意义（P＜0.05）。(4)中性单细胞凝胶电泳结果显示，彗星拖尾率、尾长、Olive尾距、尾部DNA百分含量随着DMF染毒浓度的增加而增大，经SNK方差分析，各染毒浓度组和同组阴性组比较，差异均有统计学意义（P＜0.05）。（5）经流式细胞仪检测，随着DMF染毒浓度的升高，活性氧（ROS）含量相应增高并呈线性相关关系。结论：（1）DMF能够明显抑制V79细胞增殖，存在正向的剂量-效应关系，并能引起DNA损伤。（2）DMF染毒可以引起细胞周期阻滞，导致细胞凋亡。（3）低浓度的DMF0.5mmol/L染毒6h即可引起细胞单链断裂。（4）DMF染毒后可造成细胞双链断裂，导致DNA损伤。（5）DMF可引起V79细胞内活性氧含量增加。本次研究结果显示，DMF可引起体外培养的V79细胞存活率下降，通过引起DNA单双链断裂导致细胞损伤，细胞内活性氧ROS含量的增加可能是导致DNA损伤的一种机制。
[Abstract]:Objective: dimethylformamide N, N-dimethylformamide (DMF) is a low toxic organic solvent and an important chemical raw material. It is widely used in organic synthesis, petroleum refining and other industries, as well as medicine and other industries.With the development of modern industry, the application scope of DMF is expanding, and the number of occupational contact personnel is increasing, which brings health and safety hidden trouble to workers exposed to DMF.The purpose of this study was to investigate the cytotoxic DNA damage and cytotoxic mechanism of dimethylformamide (DMF) on Chinese hamster lung cells (V79 cells), in order to explore the possible cellular damage mechanism of dimethylformamide.To provide the basis for effective measures to protect the health of occupational exposed population.Methods V79 cells were cultured in vitro. The cell survival rate of V79 cells treated with different concentrations of DMF was determined by MTT assay.To understand the relationship between concentration, time and toxicity, we observed the distribution of cell cycle by pyridine iodide (PI) staining.To investigate the effect of DMF on the cell cycle of V79 cells: (1) Detection of DNA single strand breaks in V79 cells with different concentrations of DMF at different concentrations by alkaline single cell gel electrophoresis (BSCGE). (4) Neutral single cell gel electrophoresis (neutrophilic single cell gel electrophoresis) was used to detect the damage of DNA in V79 cells after exposure to different concentrations of DMF for different periods of time.The double strand breaks of DNA in V79 cells with different concentrations of DMF were measured. The changes of Ros content in V79 cells treated with different concentrations of DMF were detected by flow cytometry DCFH-DA method.Results MTT showed that the survival rate of V79 cells decreased with the increase of the concentration and time of exposure.There was a significant dose-effect relationship for 6 h, 尾 -0.002 (P < 0.05), 尾 -0.003 (P < 0.05), 尾 -0.003 (P < 0.05) and 尾 -0.003 (P < 0.05). Flow cytometry (FCM) showed that V79 cell cycle changed significantly after 24 hours of DMF treatment, and there was a significant difference between the two groups (P < 0.05, P < 0.05, P < 0.05), and there was a significant dose-effect relationship between the two groups for 6 h, 尾 -0.002 (P < 0.05), 尾 -0.003 (P < 0.05) and 尾 -0.003 (P < 0.05).The ratio of G 1 phase cells to G 2 phase cells decreased with the increase of DMF concentration, while the proportion of S phase cells and G 2 phase cells increased with the increase of DMF concentration.The difference was statistically significant (P < 0.05). The results of alkaline single-cell gel electrophoresis showed that comet tail rate, tail length Olive tail distance and tail DNA content increased with the increase of DMF concentration.The results of neutrophil gel electrophoresis showed that comet tail rate, tail length Olive tail distance and tail DNA content increased with the increase of DMF concentration.By SNK variance analysis, the difference was statistically significant (P < 0.05) by flow cytometry. With the increase of DMF concentration, the content of reactive oxygen species (Ros) increased and showed a linear correlation.Conclusion the proliferation of V79 cells was significantly inhibited by the presence of a positive dose-effect relationship, and the cell cycle arrest could be induced by DNA damage.Low concentration of DMF0.5mmol/L could induce cell single strand break and double strand breakage after exposure to DMF0.5mmol/L for 6 h, and lead to DNA damage and increase the content of reactive oxygen species in V79 cells.The results showed that the survival rate of V79 cells in vitro was decreased. The increase of reactive oxygen species (ROS) content in V79 cells may be a possible mechanism of DNA damage by inducing single and double strand breaks of DNA.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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