二甲基甲酰胺對心肌細(xì)胞毒性及其氧化應(yīng)激機(jī)制研究
發(fā)布時間：2018-08-23 09:35
【摘要】：目的探討二甲基甲酰胺(DMF)對小鼠心肌細(xì)胞(H9c2)的細(xì)胞毒性和氧化應(yīng)激在其細(xì)胞毒性中的作用及VitC的保護(hù)效應(yīng)。 方法采用CCK-8法檢測不同劑量梯度（0mM，50mM，100mM，150mM，200mM，250mM，300mM）DMF作用心肌細(xì)胞24h、48h、72h后的細(xì)胞毒性，并用AnnexinV-FITC/PI流式細(xì)胞術(shù)檢測細(xì)胞凋亡率，用ROS、T-AOC、MDA、SOD、GSH試劑盒檢測DMF作用后細(xì)胞的氧化應(yīng)激狀態(tài)。同時DMF（100mM）作用24h、48h、72h后，用0.025mM、0.05mM、0.10mM、0.25mM的VitC處理，以觀察VitC的保護(hù)效應(yīng)。 結(jié)果不同濃度的DMF分別作用于心肌細(xì)胞24h、48h和72h，產(chǎn)生了明顯的細(xì)胞毒性,并呈現(xiàn)顯著的劑量-時間效應(yīng)關(guān)系。用DMF對心肌細(xì)胞進(jìn)行染毒后細(xì)胞發(fā)生了脂質(zhì)過氧化,并呈現(xiàn)一定的劑量-時間效應(yīng)關(guān)系。用CCK-8法檢測DMF作用于心肌細(xì)胞的24hIC50是250mM，48hIC50是220mM，72hIC50是200mM。在氧化應(yīng)激指標(biāo)的測量中，與溶劑對照組相比,DMF染毒24h（100mM，140mM，180mM，220mM，250mM）組,48h（100mM，140mM，180mM，200mM，，220mM）組和72h（100mM，125mM，140mM，180mM，200mM）組ROS升高（P0.05），DMF染毒48h（220mM）組和72h（140mM、180mM）組分別與24h組相應(yīng)濃度相比ROS升高（P0.05），DMF染毒72h（140mM、200mM）組與48h組相應(yīng)濃度相比ROS升高（P0.05）；與溶劑對照組相比, DMF染毒24h（220mM、250mM）組,48h（180mM、200mM、220mM）組和72h（100mM、125mM、140mM、180mM、200mM）組T-AOC含量減少（P0.05），DMF染毒48h（220mM）組和72h（140mM、180mM、200mM）組分別與24h組相應(yīng)濃度相比T-AOC含量減少（P0.05）， DMF染毒72h（140mM、180mM、200mM）組與48h組相應(yīng)濃度相比T-AOC含量減少（P0.05）；與溶劑對照組相比, DMF染毒24h（180mM、220mM、250mM）組,48h（140mM、180mM、200mM、220mM）組和72h（100mM、125mM、140mM、180mM、200mM）組SOD含量降低（P0.05），DMF染毒48h（220mM）組和72h（100mM、140mM、180mM）組分別與24h組相應(yīng)濃度相比SOD含量降低（P0.05），DMF染毒72h（140mM、180mM、200mM）組與48h組相應(yīng)濃度相比SOD含量降低（P0.05）；與溶劑對照組相比, DMF染毒24h（180mM、220mM、250mM）組,48h（140mM、180mM、200mM、220mM）組和72h（100mM、125mM、140mM、180mM、200mM）組GSH含量下降（P0.05），DMF染毒48h（220mM）組和72h（140mM、180mM）組分別與24h組相應(yīng)濃度相比GSH含量下降（P0.05），DMF染毒72h（140mM、180mM、200mM）組48h組相應(yīng)濃度相比GSH含量下降（P0.05）；與溶劑對照組相比, DMF染毒24h（180mM、220mM、250mM）組,48h（140mM、180mM、200mM、220mM）組和72h（125mM、140mM、180mM、200mM）組MDA含量升高（P0.05），DMF染毒72h（140mM、200mM）組與24h組相應(yīng)濃度相比MDA含量升高（P0.05），DMF染毒72h（140mM、200mM）組與48h組相應(yīng)濃度相比MDA含量升高（P0.05）。保護(hù)組VitC在低濃度（0.025mM、0.05mM、0.10mM）時對DMF對心肌細(xì)胞的氧化損傷具有保護(hù)作用，使ROS降低，T-AOC含量增多， SOD含量升高，GSH含量升高，MDA含量減少，差異具有統(tǒng)計學(xué)意義(P0.05)。保護(hù)組VitC在高濃度（0.25mM）時反而加重DMF對心肌細(xì)胞的氧化損傷，差異具有統(tǒng)計學(xué)意義(P0.05)。通過DMF對心肌細(xì)胞的氧化應(yīng)激檢測指標(biāo)（ROS、T-AOC、SOD、GSH、MDA）與濃度和時間的都具有相關(guān)性分析（P＜0.05）。可以說明，在相同作用時間時隨著DMF濃度的增加心肌細(xì)胞的氧化應(yīng)激加重，相同濃度時隨著DMF作用時間的延長心肌細(xì)胞的氧化應(yīng)激也加重。 結(jié)論二甲基甲酰胺(DMF)對小鼠心肌細(xì)胞細(xì)胞(H9c2)產(chǎn)生了明顯的細(xì)胞毒性和氧化損傷,并呈現(xiàn)一定的劑量-時間效應(yīng)關(guān)系。DMF引起了心肌細(xì)胞的氧化應(yīng)激反應(yīng)，使心肌細(xì)胞的ROS升高、T-AOC含量減少、SOD含量降低、GSH含量下降、MDA含量升高，從而證實DMF所致的氧化應(yīng)激是其引起細(xì)胞毒性的重要機(jī)制。DMF對心肌細(xì)胞的氧化應(yīng)激檢測指標(biāo)（ROS、T-AOC、SOD、GSH、MDA）與濃度和時間的相關(guān)，進(jìn)一步說明證實DMF所致的氧化應(yīng)激是其引起細(xì)胞毒性的重要機(jī)制。小劑量VitC（0.025mM、0.05mM、0.10mM）對DMF致心肌細(xì)胞的氧化損傷具有明顯的保護(hù)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R114
本文編號：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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R114
【共引文獻(xiàn)】
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