二甲基甲酰胺对心肌细胞毒性及其氧化应激机制研究
发布时间：2018-08-23 09:35
【摘要】：目的探讨二甲基甲酰胺(DMF)对小鼠心肌细胞(H9c2)的细胞毒性和氧化应激在其细胞毒性中的作用及VitC的保护效应。 方法采用CCK-8法检测不同剂量梯度（0mM，50mM，100mM，150mM，200mM，250mM，300mM）DMF作用心肌细胞24h、48h、72h后的细胞毒性，并用AnnexinV-FITC/PI流式细胞术检测细胞凋亡率，用ROS、T-AOC、MDA、SOD、GSH试剂盒检测DMF作用后细胞的氧化应激状态。同时DMF（100mM）作用24h、48h、72h后，用0.025mM、0.05mM、0.10mM、0.25mM的VitC处理，以观察VitC的保护效应。 结果不同浓度的DMF分别作用于心肌细胞24h、48h和72h，产生了明显的细胞毒性,并呈现显著的剂量-时间效应关系。用DMF对心肌细胞进行染毒后细胞发生了脂质过氧化,并呈现一定的剂量-时间效应关系。用CCK-8法检测DMF作用于心肌细胞的24hIC50是250mM，48hIC50是220mM，72hIC50是200mM。在氧化应激指标的测量中，与溶剂对照组相比,DMF染毒24h（100mM，140mM，180mM，220mM，250mM）组,48h（100mM，140mM，180mM，200mM，，220mM）组和72h（100mM，125mM，140mM，180mM，200mM）组ROS升高（P0.05），DMF染毒48h（220mM）组和72h（140mM、180mM）组分别与24h组相应浓度相比ROS升高（P0.05），DMF染毒72h（140mM、200mM）组与48h组相应浓度相比ROS升高（P0.05）；与溶剂对照组相比, DMF染毒24h（220mM、250mM）组,48h（180mM、200mM、220mM）组和72h（100mM、125mM、140mM、180mM、200mM）组T-AOC含量减少（P0.05），DMF染毒48h（220mM）组和72h（140mM、180mM、200mM）组分别与24h组相应浓度相比T-AOC含量减少（P0.05）， DMF染毒72h（140mM、180mM、200mM）组与48h组相应浓度相比T-AOC含量减少（P0.05）；与溶剂对照组相比, DMF染毒24h（180mM、220mM、250mM）组,48h（140mM、180mM、200mM、220mM）组和72h（100mM、125mM、140mM、180mM、200mM）组SOD含量降低（P0.05），DMF染毒48h（220mM）组和72h（100mM、140mM、180mM）组分别与24h组相应浓度相比SOD含量降低（P0.05），DMF染毒72h（140mM、180mM、200mM）组与48h组相应浓度相比SOD含量降低（P0.05）；与溶剂对照组相比, DMF染毒24h（180mM、220mM、250mM）组,48h（140mM、180mM、200mM、220mM）组和72h（100mM、125mM、140mM、180mM、200mM）组GSH含量下降（P0.05），DMF染毒48h（220mM）组和72h（140mM、180mM）组分别与24h组相应浓度相比GSH含量下降（P0.05），DMF染毒72h（140mM、180mM、200mM）组48h组相应浓度相比GSH含量下降（P0.05）；与溶剂对照组相比, DMF染毒24h（180mM、220mM、250mM）组,48h（140mM、180mM、200mM、220mM）组和72h（125mM、140mM、180mM、200mM）组MDA含量升高（P0.05），DMF染毒72h（140mM、200mM）组与24h组相应浓度相比MDA含量升高（P0.05），DMF染毒72h（140mM、200mM）组与48h组相应浓度相比MDA含量升高（P0.05）。保护组VitC在低浓度（0.025mM、0.05mM、0.10mM）时对DMF对心肌细胞的氧化损伤具有保护作用，使ROS降低，T-AOC含量增多， SOD含量升高，GSH含量升高，MDA含量减少，差异具有统计学意义(P0.05)。保护组VitC在高浓度（0.25mM）时反而加重DMF对心肌细胞的氧化损伤，差异具有统计学意义(P0.05)。通过DMF对心肌细胞的氧化应激检测指标（ROS、T-AOC、SOD、GSH、MDA）与浓度和时间的都具有相关性分析（P＜0.05）。可以说明，在相同作用时间时随着DMF浓度的增加心肌细胞的氧化应激加重，相同浓度时随着DMF作用时间的延长心肌细胞的氧化应激也加重。 结论二甲基甲酰胺(DMF)对小鼠心肌细胞细胞(H9c2)产生了明显的细胞毒性和氧化损伤,并呈现一定的剂量-时间效应关系。DMF引起了心肌细胞的氧化应激反应，使心肌细胞的ROS升高、T-AOC含量减少、SOD含量降低、GSH含量下降、MDA含量升高，从而证实DMF所致的氧化应激是其引起细胞毒性的重要机制。DMF对心肌细胞的氧化应激检测指标（ROS、T-AOC、SOD、GSH、MDA）与浓度和时间的相关，进一步说明证实DMF所致的氧化应激是其引起细胞毒性的重要机制。小剂量VitC（0.025mM、0.05mM、0.10mM）对DMF致心肌细胞的氧化损伤具有明显的保护作用。
[Abstract]:Objective To investigate the cytotoxicity of dimethylformamide (DMF) on mouse cardiomyocytes (H9c2) and the role of oxidative stress in its cytotoxicity and the protective effect of VitC.
Methods CCK-8 assay was used to detect the cytotoxicity of different dosage gradients (0 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM) DMF treated cardiomyocytes 24, 48, 72 hours later. Annexin V-FITC/PI flow cytometry was used to detect the apoptosis rate. ROS, T-AOC, MDA, SOD, GSH kits were used to detect the oxidative stress state of the cells after DMF (100 mM) treatment for 24 hours. After 48h, 72h was processed with VitC of 0.025mM, 0.05mM, 0.10mM and 0.25mM to observe the protective effect of VitC.
Results Different concentrations of DMF had significant cytotoxicity and dose-time effect on myocardial cells at 24h, 48h and 72h, respectively. Lipid peroxidation occurred in myocardial cells after exposure to DMF, and there was a dose-time effect relationship. The 24hI of DMF on myocardial cells was detected by CCK-8 method. C50 is 250mM, 48hIC50 is 220 mM, 4848hIC50 is 220 mM, 7272hIC50 is 200 mM. In the measurement of oxidstress index, compared with the control group, DMF exposure 24h (100 mM, 140 mM, 180 mM, 220 mM, 250250mM) group, 48h (100 mM, 140 mM, 140 mM, 180 180 mM, 180 180 mM, 180 mM, 200 mM, 200 mM, 220 mM, 220mM) group and 72h (100 mM, 125mM, 125mM, 140 mM, 140 140 mM, 180 mM, 180 mM, 180 m, 180 mM, 180 mM, 200 200 200 mM, 200 200 200 200 mM) group and 72h (100, DMDMDMM, 120, 48hours, 48Groups 24 Compared with the control group, the levels of ROS in DMF treated group were higher at 72h (140 mM, 200 mM) and 48h (220 mM, 250 mM), 48h (180 mM, 200 mM) and 72h (100 mM, 125 mM, 140 mM, 180 mM, 200 mM) respectively (P The content of T-AOC in 200 mM group was lower than that in 24 h group (P 0.05). The content of T-AOC in 72 h group (140 mM, 180 mM, 200 mM) was lower than that in 48 h group (P 0.05). Compared with solvent control group, the content of SOD in 24 h group (180 mM, 220 mM, 250 mM) and 48 h group (140 mM, 180 mM, 200 mM, 220 mM) and 72 h group (100 mM, 140 mM, 140 mM, 180 mM, 200 mM) were lower than that in 48 h group (100 mM, 180 mM, 200 mM, 200 mM). Compared with the corresponding concentration of 24 h group (P 0.05), the SOD content of 48 h (220 mM) group and 72 h (100 mM, 140 mM, 180 mM) group was lower (P 0.05), and that of 72 h (140 mM, 180 mM, 200 mM) group and 48 h (180 mM, 180 mM, 200 mM) group was lower (P 0.05); compared with the solvent control group, the SOD content of 24 h (180 mM, 220 mM, 250 mM) group, 48 h (140 mM, 180 mM, 200 mM) group was lower (P 0.05). GSH content decreased (P 0.05) in 72h (100 mM, 125 mM, 140 mM, 180 mM, 200 mM) group, 48h (220 mM) group and 72h (140 mM, 180 mM) group compared with the corresponding concentration in 24h group (P 0.05), and GSH content decreased (P 0.05) in 72h (140 mM, 180 mM, 200 mM) group compared with that in solvent control group (P 0.05). MDA content in 0 mM group, 48h (140 mM, 180 mM, 200 mM, 220 mM) group and 72h (125 mM, 140 mM, 180 mM, 200 mM) group increased (P 0.05), and the MDA content in 72h (140 mM, 200 mM) group was higher than that in 24h group (P 0.05), and the MDA content in 72h (140 mM, 200 mM) group was higher than that in 48h group (P 0.05). (P 0.05). VitC in the protective group increased the oxidative damage of DMF to myocardial cells at high concentration (0.25 mM), but increased the oxidative damage of DMF to myocardial cells. The difference was statistically significant (P 0.05). The correlation between the oxidative stress index (ROS, T-AOC, SOD, GSH, MDA) and the concentration and time was analyzed (P < 0.05).
Conclusion Dimethylformamide (DMF) has obvious cytotoxicity and oxidative damage to mouse cardiomyocytes (H9c2) in a dose-time dependent manner. DMF induces oxidative stress in cardiomyocytes, which increases ROS, T-AOC content, SOD content, GSH content and MDA content in cardiomyocytes. It was confirmed that oxidative stress induced by DMF was an important mechanism of cytotoxicity. The oxidative stress index (ROS, T-AOC, SOD, GSH, MDA) of DMF on myocardial cells was correlated with concentration and time. It was further confirmed that oxidative stress induced by DMF was an important mechanism of cytotoxicity. The oxidative damage of muscle cells has an obvious protective effect.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114
本文编号：2198647
[Abstract]:Objective To investigate the cytotoxicity of dimethylformamide (DMF) on mouse cardiomyocytes (H9c2) and the role of oxidative stress in its cytotoxicity and the protective effect of VitC.
Methods CCK-8 assay was used to detect the cytotoxicity of different dosage gradients (0 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM) DMF treated cardiomyocytes 24, 48, 72 hours later. Annexin V-FITC/PI flow cytometry was used to detect the apoptosis rate. ROS, T-AOC, MDA, SOD, GSH kits were used to detect the oxidative stress state of the cells after DMF (100 mM) treatment for 24 hours. After 48h, 72h was processed with VitC of 0.025mM, 0.05mM, 0.10mM and 0.25mM to observe the protective effect of VitC.
Results Different concentrations of DMF had significant cytotoxicity and dose-time effect on myocardial cells at 24h, 48h and 72h, respectively. Lipid peroxidation occurred in myocardial cells after exposure to DMF, and there was a dose-time effect relationship. The 24hI of DMF on myocardial cells was detected by CCK-8 method. C50 is 250mM, 48hIC50 is 220 mM, 4848hIC50 is 220 mM, 7272hIC50 is 200 mM. In the measurement of oxidstress index, compared with the control group, DMF exposure 24h (100 mM, 140 mM, 180 mM, 220 mM, 250250mM) group, 48h (100 mM, 140 mM, 140 mM, 180 180 mM, 180 180 mM, 180 mM, 200 mM, 200 mM, 220 mM, 220mM) group and 72h (100 mM, 125mM, 125mM, 140 mM, 140 140 mM, 180 mM, 180 mM, 180 m, 180 mM, 180 mM, 200 200 200 mM, 200 200 200 200 mM) group and 72h (100, DMDMDMM, 120, 48hours, 48Groups 24 Compared with the control group, the levels of ROS in DMF treated group were higher at 72h (140 mM, 200 mM) and 48h (220 mM, 250 mM), 48h (180 mM, 200 mM) and 72h (100 mM, 125 mM, 140 mM, 180 mM, 200 mM) respectively (P The content of T-AOC in 200 mM group was lower than that in 24 h group (P 0.05). The content of T-AOC in 72 h group (140 mM, 180 mM, 200 mM) was lower than that in 48 h group (P 0.05). Compared with solvent control group, the content of SOD in 24 h group (180 mM, 220 mM, 250 mM) and 48 h group (140 mM, 180 mM, 200 mM, 220 mM) and 72 h group (100 mM, 140 mM, 140 mM, 180 mM, 200 mM) were lower than that in 48 h group (100 mM, 180 mM, 200 mM, 200 mM). Compared with the corresponding concentration of 24 h group (P 0.05), the SOD content of 48 h (220 mM) group and 72 h (100 mM, 140 mM, 180 mM) group was lower (P 0.05), and that of 72 h (140 mM, 180 mM, 200 mM) group and 48 h (180 mM, 180 mM, 200 mM) group was lower (P 0.05); compared with the solvent control group, the SOD content of 24 h (180 mM, 220 mM, 250 mM) group, 48 h (140 mM, 180 mM, 200 mM) group was lower (P 0.05). GSH content decreased (P 0.05) in 72h (100 mM, 125 mM, 140 mM, 180 mM, 200 mM) group, 48h (220 mM) group and 72h (140 mM, 180 mM) group compared with the corresponding concentration in 24h group (P 0.05), and GSH content decreased (P 0.05) in 72h (140 mM, 180 mM, 200 mM) group compared with that in solvent control group (P 0.05). MDA content in 0 mM group, 48h (140 mM, 180 mM, 200 mM, 220 mM) group and 72h (125 mM, 140 mM, 180 mM, 200 mM) group increased (P 0.05), and the MDA content in 72h (140 mM, 200 mM) group was higher than that in 24h group (P 0.05), and the MDA content in 72h (140 mM, 200 mM) group was higher than that in 48h group (P 0.05). (P 0.05). VitC in the protective group increased the oxidative damage of DMF to myocardial cells at high concentration (0.25 mM), but increased the oxidative damage of DMF to myocardial cells. The difference was statistically significant (P 0.05). The correlation between the oxidative stress index (ROS, T-AOC, SOD, GSH, MDA) and the concentration and time was analyzed (P < 0.05).
Conclusion Dimethylformamide (DMF) has obvious cytotoxicity and oxidative damage to mouse cardiomyocytes (H9c2) in a dose-time dependent manner. DMF induces oxidative stress in cardiomyocytes, which increases ROS, T-AOC content, SOD content, GSH content and MDA content in cardiomyocytes. It was confirmed that oxidative stress induced by DMF was an important mechanism of cytotoxicity. The oxidative stress index (ROS, T-AOC, SOD, GSH, MDA) of DMF on myocardial cells was correlated with concentration and time. It was further confirmed that oxidative stress induced by DMF was an important mechanism of cytotoxicity. The oxidative damage of muscle cells has an obvious protective effect.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114
【共引文献】
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