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目的探讨乙醇及其代谢产物对心肌祖细胞的毒性作用及H3K9表突变作用。方法以心肌祖细胞株为研究对象,分别用不同剂量乙醇(200、100、50mmol/L)、乙醛(16、12、8、4mmol/L)和乙酸(16、12、8、4mmol/L)处理。应用MTT实验筛选出乙醇及其代谢产物的干预浓度,Westernblot检测组蛋白H3K9乙酰化作用,实时定量PCR检测心脏发育相关基因GATA4、Mef2c、Tbx5mRNA表达量的变化。结果 MTT结果显示低浓度组50mmol/L乙醇、4mmol/L乙醛、4mmol/L乙酸的细胞活力与对照组比较差异无统计学意义(P>0.05),不影响心肌祖细胞增殖;高浓度组200mmol/L乙醇、12mmol/L乙醛、16mmol/L乙酸对心肌祖细胞抑制率分别为31.5%、36.0%、32.1%,抑制细胞增殖(P<0.05)。低浓度组乙醇、乙酸分别使组蛋白H3K9乙酰化水平升高2.4、2.2倍(P<0.05)。心脏发育相关基因表达无明显变化(P>0.05);高浓度组乙醇、乙酸分别使组蛋白H3K9乙酰化水平升高5.3、5.6倍,同时心脏发育相关基因GATA4、Mef2c表达均增加,与对照组及相应低浓度组比较均有统计学差异(P<0.05);乙醛则无论低浓度还是高浓度对组蛋白H3K9乙酰化水平及基因表达均无明显影响(P>0.05)。结论高浓度的乙醇及其代谢产物对心肌祖细胞均有毒性作用,而乙醇及乙酸具有组蛋白H3K9表突变作用,可能为酒精致先心病发病机制之一。
Objective To investigate the toxic effects of ethanol and its metabolites on cardiac progenitor cells and the role of H3K9 in the mutation. Methods Myocardial progenitor cell lines were used as experimental subjects and were treated with different doses of ethanol (200,100,50mmol / L), acetaldehyde (16,12,8,4mmol / L) and acetic acid (16,12,8,4mmol / L )deal with. The concentrations of ethanol and its metabolites were screened by MTT assay. The histone H3K9 acetylation was detected by Western blot and the expression of GATA4, Mef2c and Tbx5 mRNA were detected by real-time quantitative PCR. Results MTT results showed that the cell viability of 50 mmol / L ethanol, 4 mmol / L acetaldehyde and 4 mmol / L acetic acid in the low concentration group was not significantly different from that in the control group (P> 0.05), but not in the high concentration group The inhibition rate of myocardial progenitor cells were 31.5%, 36.0% and 32.1% respectively with 200mmol / L ethanol, 12mmol / L acetaldehyde and 16mmol / L acetic acid, and inhibited the cell proliferation (P <0.05). Low concentrations of ethanol and acetic acid increased histone H3K9 acetylation levels 2.4 and 2.2 times (P <0.05), respectively. (P> 0.05). High concentrations of ethanol and acetic acid increased the histone H3K9 acetylation levels by 5.3 and 5.6 times, respectively. Meanwhile, the expressions of cardiac development related genes GATA4 and Mef2c were increased, which were significantly higher than those of the control group (P <0.05). Acetaldehyde had no significant effect on histone H3K9 acetylation and gene expression in both low and high concentration groups (P> 0.05). Conclusions High concentrations of ethanol and its metabolites have toxic effects on cardiac progenitor cells. However, ethanol and acetic acid have the histone H3K9 mutation, which may be one of the pathogenesis of alcohol induced CHD.