二甲基甲酰胺致H9c2心肌细胞膜损伤研究

发布时间：2018-05-19 06:02

本文选题：二甲基甲酰胺 + H9c2心肌细胞　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的:探讨二甲基甲酰胺(DMF)对H9c2心肌细胞膜损伤的影响及其可能的机制。方法:取体外培养H9c2心肌细胞,以浓度为0、25、50、75、100、125、150、175、200 mmol/L DMF处理24 h后,采用CCK-8法检测细胞存活率。根据CCK-8实验结果,以细胞存活率大于70%为原则,为观察剂量-效应关系,设立对照组和80、110、140 mmol/L组,分别给予浓度为0、80、110、140 mmol/L DMF染毒处理24h;为观察时间-效应关系,设立对照组和2、6、12、24 h组,给予浓度为110 mmol/L DMF分别染毒处理0、2、6、12、24 h。比色法检测培养上清LDH活性反映细胞膜完整性,荧光偏振法检测细胞膜荧光偏振度反映细胞膜流动性,流式细胞术检测细胞FDA+/PI+率反映细胞膜通透性,荧光法检测细胞膜胆固醇浓度,钙离子成像技术检测细胞内Ca~(2+)浓度。结果:与对照组比较,100、125、150、175、200 mmol/L组细胞存活率均降低(P0.01),存在剂量-效应关系(P0.01)。与对照组比较,80、110、140 mmol/L组LDH活性均升高(P0.01),2、6、12 h组LDH活性均升高(P0.05),存在剂量-效应关系(P0.01)和时间-效应关系(P0.01)。与对照组比较,80、110、140 mmol/L组荧光偏振度均升高(P0.01),6、12、24 h组荧光偏振度均升高(P0.01),存在剂量-效应关系(P0.01)和时间-效应关系(P0.01)。与对照组比较,80、110 mmol/L组FDA+/PI+率均升高(P0.01),6、12、24 h组FDA+/PI+率均升高(P0.05),存在时间-效应关系(P0.01);与对照组比较,110、140 mmol/L组PI+率均升高(P0.01),12、24 h组PI+率均升高(P0.05),存在剂量-效应关系(P0.01)和时间-效应关系(P0.01)。与对照组比较,80、110、140 mmol/L组胆固醇浓度均升高(P0.01),6、24 h组胆固醇浓度均升高(P0.05),存在剂量-效应关系(P0.01)和时间-效应关系(P0.01)。与对照组比较,80、110、140 mmol/L组Ca~(2+)浓度均升高(P0.01),2、6、12、24 h组Ca~(2+)均升高(P0.05),存在剂量-效应关系(P0.01)和时间-效应关系(P0.01)。在DMF染毒浓度为0~200 mmol/L时,LDH活性与荧光偏振度呈正相关(r=0.97,P0.01),与胆固醇浓度呈正相关(r=0.94,P0.01),与Ca~(2+)浓度呈正相关(r=0.68,P0.05);在DMF染毒时间为0~24 h时,LDH活性与荧光偏振度呈正相关(r=0.89,P0.01),与FDA+/PI+率呈正相关(r=0.95,P0.01),与胆固醇浓度呈正相关(r=0.88,P0.01),与Ca~(2+)浓度呈正相关(r=0.66,P0.05)。结论:二甲基甲酰胺对H9c2心肌细胞膜造成损伤;膜流动性降低、膜通透性增加、膜胆固醇稳态失衡以及细胞内钙超载参与损伤过程。
[Abstract]:Aim: to investigate the effect of dimethylformamide (DMF) on myocardial cell membrane damage in H9c2 and its possible mechanism. Methods: H9c2 cardiomyocytes were cultured in vitro. The survival rate was measured by CCK-8 method after 24 hours of treatment with the concentration of 0.2550,75100125150175200 mmol/L DMF. According to the results of CCK-8 experiment, the survival rate of cells was more than 70%. In order to observe the dose-effect relationship, the control group and the 80110140 mmol/L group were treated with 0.80110140 mmol/L DMF for 24 h, respectively, and the time-effect relationship was observed. The control group and the control group were divided into two groups: the control group and the control group. The control group and the control group were treated for 24 hours with the concentration of 110 mmol/L DMF. The activity of LDH in culture supernatant was measured by colorimetry to reflect the integrity of cell membrane, the degree of fluorescence polarization of cell membrane by fluorescence polarization method reflected the membrane fluidity, and the rate of FDA / Pi by flow cytometry reflected the permeability of cell membrane. The concentration of plasma membrane cholesterol and intracellular Ca~(2 were detected by fluorescence method and calcium imaging technique. Results: compared with the control group, the cell survival rate of the 100125 150175200 mmol/L group was significantly lower than that of the control group, and there was a dose-effect relationship between the two groups. Compared with the control group, the activity of LDH in the 80110140 mmol/L group was significantly higher than that in the control group. The LDH activity in the control group was significantly higher than that in the control group for 12 h. There was a dose-effect relationship (P0.01) and a time-response relationship (P0.01a). Compared with the control group, the fluorescence polarization degree of the 80110140 mmol/L group was higher than that of the control group. The fluorescence polarization degree of the control group was higher than that of the control group for 24 h. There was a dose-effect relationship (P0.01) and a time-effect relationship (P0.01). Compared with the control group, the ratio of FDA / Pi in the mmol/L group was significantly higher than that in the control group. The FDA / Pi ratio in the control group was significantly higher than that in the control group for 24 h. The FDA / Pi ratio in the control group was significantly higher than that in the control group (P 0.05%) and the time effect relationship was P 0.01 (P 0.01) and the time effect relationship (P 0.01), respectively, compared with the control group, and the Pi rate of the 110140 mmol/L group was significantly higher than that of the control group at 1224 h. Compared with the control group, the cholesterol concentrations of the 80110140 mmol/L group were all higher than that of the control group. The cholesterol concentrations of the control group were all higher than that of the control group for 24 h. There was a dose-effect relationship (P0.01) and a time-effect relationship (P0.01a). Compared with the control group, the concentration of Ca~(2 in the 80110140 mmol/L group was significantly higher than that in the control group (P 0.01) and the Ca~(2 in the control group was significantly higher than that in the control group. There was a dose-effect relationship (P 0.01) and a time-effect relationship (P 0.01). There was a positive correlation between the activity of DMF and the degree of fluorescence polarization at the concentration of 0 ~ 200 mmol/L, a positive correlation between the activity of DMF and the degree of fluorescence polarization, a positive correlation between the activity of DMF with the concentration of cholesterol, a positive correlation with the concentration of Ca~(2, and a positive correlation between the activity of DMF and the degree of fluorescence polarization, and a positive correlation between the activity of DMF and the degree of fluorescence polarization. The ratio of FDA / Pi was positively correlated with the concentration of cholesterol and Ca~(2. Conclusion: dimethylformamide can damage H9c2 myocardial cell membrane, membrane fluidity decreases, membrane permeability increases, membrane cholesterol homeostasis and intracellular calcium overload participate in the damage process.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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