發(fā)布時(shí)間：2018-05-19 06:02

  本文選題：二甲基甲酰胺 + H9c2心肌細(xì)胞�。� 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討二甲基甲酰胺(DMF)對(duì)H9c2心肌細(xì)胞膜損傷的影響及其可能的機(jī)制。方法:取體外培養(yǎng)H9c2心肌細(xì)胞,以濃度為0、25、50、75、100、125、150、175、200 mmol/L DMF處理24 h后,采用CCK-8法檢測(cè)細(xì)胞存活率。根據(jù)CCK-8實(shí)驗(yàn)結(jié)果,以細(xì)胞存活率大于70%為原則,為觀(guān)察劑量-效應(yīng)關(guān)系,設(shè)立對(duì)照組和80、110、140 mmol/L組,分別給予濃度為0、80、110、140 mmol/L DMF染毒處理24h;為觀(guān)察時(shí)間-效應(yīng)關(guān)系,設(shè)立對(duì)照組和2、6、12、24 h組,給予濃度為110 mmol/L DMF分別染毒處理0、2、6、12、24 h。比色法檢測(cè)培養(yǎng)上清LDH活性反映細(xì)胞膜完整性,熒光偏振法檢測(cè)細(xì)胞膜熒光偏振度反映細(xì)胞膜流動(dòng)性,流式細(xì)胞術(shù)檢測(cè)細(xì)胞FDA+/PI+率反映細(xì)胞膜通透性,熒光法檢測(cè)細(xì)胞膜膽固醇濃度,鈣離子成像技術(shù)檢測(cè)細(xì)胞內(nèi)Ca~(2+)濃度。結(jié)果:與對(duì)照組比較,100、125、150、175、200 mmol/L組細(xì)胞存活率均降低(P0.01),存在劑量-效應(yīng)關(guān)系(P0.01)。與對(duì)照組比較,80、110、140 mmol/L組LDH活性均升高(P0.01),2、6、12 h組LDH活性均升高(P0.05),存在劑量-效應(yīng)關(guān)系(P0.01)和時(shí)間-效應(yīng)關(guān)系(P0.01)。與對(duì)照組比較,80、110、140 mmol/L組熒光偏振度均升高(P0.01),6、12、24 h組熒光偏振度均升高(P0.01),存在劑量-效應(yīng)關(guān)系(P0.01)和時(shí)間-效應(yīng)關(guān)系(P0.01)。與對(duì)照組比較,80、110 mmol/L組FDA+/PI+率均升高(P0.01),6、12、24 h組FDA+/PI+率均升高(P0.05),存在時(shí)間-效應(yīng)關(guān)系(P0.01);與對(duì)照組比較,110、140 mmol/L組PI+率均升高(P0.01),12、24 h組PI+率均升高(P0.05),存在劑量-效應(yīng)關(guān)系(P0.01)和時(shí)間-效應(yīng)關(guān)系(P0.01)。與對(duì)照組比較,80、110、140 mmol/L組膽固醇濃度均升高(P0.01),6、24 h組膽固醇濃度均升高(P0.05),存在劑量-效應(yīng)關(guān)系(P0.01)和時(shí)間-效應(yīng)關(guān)系(P0.01)。與對(duì)照組比較,80、110、140 mmol/L組Ca~(2+)濃度均升高(P0.01),2、6、12、24 h組Ca~(2+)均升高(P0.05),存在劑量-效應(yīng)關(guān)系(P0.01)和時(shí)間-效應(yīng)關(guān)系(P0.01)。在DMF染毒濃度為0~200 mmol/L時(shí),LDH活性與熒光偏振度呈正相關(guān)(r=0.97,P0.01),與膽固醇濃度呈正相關(guān)(r=0.94,P0.01),與Ca~(2+)濃度呈正相關(guān)(r=0.68,P0.05);在DMF染毒時(shí)間為0~24 h時(shí),LDH活性與熒光偏振度呈正相關(guān)(r=0.89,P0.01),與FDA+/PI+率呈正相關(guān)(r=0.95,P0.01),與膽固醇濃度呈正相關(guān)(r=0.88,P0.01),與Ca~(2+)濃度呈正相關(guān)(r=0.66,P0.05)。結(jié)論:二甲基甲酰胺對(duì)H9c2心肌細(xì)胞膜造成損傷;膜流動(dòng)性降低、膜通透性增加、膜膽固醇穩(wěn)態(tài)失衡以及細(xì)胞內(nèi)鈣超載參與損傷過(guò)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R114

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