本文選題：二甲基甲酰胺 + MTT��； 參考：《蘇州大學》2013年碩士論文

【摘要】：目的：二甲基甲酰胺(N, N-dimethylformamide, DMF)是一種低毒類的有機溶劑，是一種重要的化工原料，目前廣泛應用于有機合成、石油提煉等工業(yè)行業(yè)，以及醫(yī)藥和其他行業(yè)。隨著現代工業(yè)的發(fā)展，DMF的應用范圍不斷擴大，職業(yè)接觸人員也不斷增加，這給接觸DMF的作業(yè)人群帶來了健康安全隱患。本研究通過了解二甲基甲酰胺（DMF）對中國倉鼠肺細胞（V79細胞）的細胞毒性、DNA損傷作用以及細胞毒性機制，試圖探討二甲基甲酰胺可能的細胞損傷機制，為提出有效的保護職業(yè)暴露人群健康的措施提供依據。 方法：（1）以體外培養(yǎng)V79細胞為研究對象，采用四氮唑鹽比色分析法（MTT法）檢測不同濃度的DMF對V79細胞作用不同時間段后，細胞存活率的變化，了解濃度-時間-毒性效應關系；（2）采用碘化吡啶（PI)染色觀察細胞周期分布狀況，了解DMF對V79細胞周期的影響；（3）用堿性單細胞凝膠電泳技術（SCGE）檢測不同濃度DMF在作用不同時間段后，V79細胞的DNA單鏈斷裂的損傷情況。（4）用中性單細胞凝膠電泳技術檢測不同濃度DMF在作用不同時間段后，V79細胞的DNA雙鏈斷裂的情況；(5)用流式細胞術DCFH-DA法檢測不同濃度DMF處理V79細胞后活性氧（ROS）含量比的變化。 結果：(1) MTT結果顯示，各組V79細胞存活率隨染毒濃度及染毒時間的增加而下降，與陰性組比較差異均有統(tǒng)計學意義（P＜0.05），存在明顯的劑量-效應關系（6h, β=-0.002，P＜0.05;12h, β=-0.003，P＜0.05;24h, β=-0.003，P＜0.05）。(2)流式細胞儀檢測細胞周期發(fā)現，經DMF作用24h后V79細胞周期發(fā)生了明顯的變化。每一染毒時間段內，，隨著DMF染毒濃度的增加，G1期細胞的構成比降低；而S期細胞和G2期細胞的比例則隨著DMF濃度的升高而升高，各染毒組與陰性對照組比較均有差異性，差異具有統(tǒng)計學意義（P＜0.05）。(3)堿性單細胞凝膠電泳結果顯示，彗星拖尾率、尾長、Olive尾距、尾部DNA百分含量隨著DMF染毒濃度的增加而增大，經SNK方差分析，各染毒濃度組和同組陰性組比較，差異均有統(tǒng)計學意義（P＜0.05）。(4)中性單細胞凝膠電泳結果顯示，彗星拖尾率、尾長、Olive尾距、尾部DNA百分含量隨著DMF染毒濃度的增加而增大，經SNK方差分析，各染毒濃度組和同組陰性組比較，差異均有統(tǒng)計學意義（P＜0.05）。（5）經流式細胞儀檢測，隨著DMF染毒濃度的升高，活性氧（ROS）含量相應增高并呈線性相關關系。 結論：（1）DMF能夠明顯抑制V79細胞增殖，存在正向的劑量-效應關系，并能引起DNA損傷。（2）DMF染毒可以引起細胞周期阻滯，導致細胞凋亡。（3）低濃度的DMF0.5mmol/L染毒6h即可引起細胞單鏈斷裂。（4）DMF染毒后可造成細胞雙鏈斷裂，導致DNA損傷。（5）DMF可引起V79細胞內活性氧含量增加。本次研究結果顯示，DMF可引起體外培養(yǎng)的V79細胞存活率下降，通過引起DNA單雙鏈斷裂導致細胞損傷，細胞內活性氧ROS含量的增加可能是導致DNA損傷的一種機制。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R114

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