乙醇及其代谢产物对心肌祖细胞的毒性及H3K9表突变作用
发布时间：2018-08-25 15:39
【摘要】： 目的: 孕期酒精暴露可导致先天性心脏病(Congenital heart disease,CHD,简称先心病),但其具体机制目前仍不清楚。我们前期研究提示组蛋白乙酰化修饰失衡可引起心脏发育相关基因表达异常(即表突变,Epimutation),可能与先心病发生有关。目前研究表明,乙醇可选择性引起体外培养肝细胞组蛋白H3第9位赖氨酸(Histone H3 lysine 9,H3K9)乙酰化修饰失衡,但是否引起心脏中的H3K9乙酰化修饰失衡以及进一步影响心脏发育相关基因的表达(即H3K9表突变)仍不清楚。本研究以心肌祖细胞15-13(Cardiac progenitor cells15-13,CP15-13)为研究对象,探讨乙醇及其代谢产物对心肌祖细胞的毒性及H3K9表突变作用。 材料与方法: 以CP15-13为研究对象,复苏细胞后,用含10%胎牛血清的DMEM高糖培养基培养,待细胞贴满瓶底80%左右时以1:3比例传代,取第四代细胞做干预实验。应用MTT比色法观察乙醇及其代谢产物对心肌祖细胞的毒性作用及筛选出低、高浓度干预组。剂量设计为乙醇50,100,200mM,乙醛及乙酸均设计为4,8,12,16mM。低、高浓度干预CP15-13后,应用Western blot方法检测CP15-13干预前后组蛋白H3K9乙酰化水平改变。应用基于SYBR GREENⅠ的荧光定量聚合酶链反应(quantitative polymerase chain reaction, Q-PCR)方法检测CP15-13心脏发育相关基因GATA4、Mef2c、Tbx5 mRNA表达量在干预前后的变化。 结果: 1.MTT结果显示50 mM乙醇(0.368±0.028)、4 mM乙醛(0.336±0.040)、4 mM乙酸(0.359±0.014)与对照组(0.358±0.066)比较不影响心肌祖细胞增殖(P0.05),作为低浓度干预组, 200 mM乙醇(0.245±0.042)、12 mM乙醛(0.220±0.017)、16 mM乙酸(0.243±0.024)与对照组(0.358±0.066)比较,对心肌祖细胞抑制率为30%左右(P0.05),作为高浓度干预组。 2.MTT筛选出低、高浓度干预CP15-13后,应用Western blot法及Q-PCR法分别对组蛋白H3K9乙酰化水平及心脏发育相关基因mRNA表达水平进行对比,结果显示低浓度组乙醇、乙酸分别使组蛋白H3K9乙酰化水平升高2.4、2.2倍(P0.05),心脏发育相关基因表达无明显变化(P0.05),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表达分别增加(1.767±0.173)、(1.518±0.133),Mef2c表达分别增加(3.301±0.465)、(1.875±0.587),与对照组及相应低浓度组比较均有统计学差异(P0.05),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(P0.05)。 结论: 1.高浓度的乙醇及其代谢产物对心肌祖细胞均有毒性作用 2.乙醇及其代谢产物之一乙酸对心肌祖细胞具有组蛋白H3K9表突变作用,而其另一代谢产物乙醛无此作用。
[Abstract]:Objective: alcohol exposure during pregnancy may lead to congenital heart disease (Congenital heart disease,CHD,), but its mechanism is still unclear. Our previous study suggested that the imbalance of acetylation modification of histone may cause abnormal expression of cardiac development-related genes (i.e. epigenetic mutation), which may be related to the occurrence of congenital heart disease. It has been shown that ethanol can selectively induce the imbalance of acetylation modification of Histone H3 lysine 9 H3K9 in cultured hepatocytes in vitro. However, it is not clear whether the imbalance of H3K9 acetylation modification in the heart and the expression of genes related to cardiac development (i.e. H3K9 epigenetic mutation) are affected further. In this study, myocardial progenitor cells (15-13 (Cardiac progenitor cells15-13,CP15-13) were used to study the toxicity of ethanol and its metabolites to myocardial progenitor cells and the mutagenesis of H3K9. Materials and methods: CP15-13 cells were resuscitated and cultured on DMEM medium containing 10% fetal bovine serum. When the cells were filled with 80% of the bottom of the bottle, the cells were subcultured at 1:3, and the fourth passage cells were taken for intervention experiment. The toxicity of ethanol and its metabolites to myocardial progenitor cells was observed by MTT colorimetry. The dose design was ethanol 50100200mMand acetaldehyde and acetic acid were designed as 40.812mM and 16mMrespectively. The acetylation level of histone H3K9 was detected by Western blot before and after CP15-13 intervention after low and high concentration of CP15-13. Fluorescence quantitative polymerase chain reaction (quantitative polymerase chain reaction, Q-PCR) based on SYBR GREEN 鈪，
本文编号：2203363
[Abstract]:Objective: alcohol exposure during pregnancy may lead to congenital heart disease (Congenital heart disease,CHD,), but its mechanism is still unclear. Our previous study suggested that the imbalance of acetylation modification of histone may cause abnormal expression of cardiac development-related genes (i.e. epigenetic mutation), which may be related to the occurrence of congenital heart disease. It has been shown that ethanol can selectively induce the imbalance of acetylation modification of Histone H3 lysine 9 H3K9 in cultured hepatocytes in vitro. However, it is not clear whether the imbalance of H3K9 acetylation modification in the heart and the expression of genes related to cardiac development (i.e. H3K9 epigenetic mutation) are affected further. In this study, myocardial progenitor cells (15-13 (Cardiac progenitor cells15-13,CP15-13) were used to study the toxicity of ethanol and its metabolites to myocardial progenitor cells and the mutagenesis of H3K9. Materials and methods: CP15-13 cells were resuscitated and cultured on DMEM medium containing 10% fetal bovine serum. When the cells were filled with 80% of the bottom of the bottle, the cells were subcultured at 1:3, and the fourth passage cells were taken for intervention experiment. The toxicity of ethanol and its metabolites to myocardial progenitor cells was observed by MTT colorimetry. The dose design was ethanol 50100200mMand acetaldehyde and acetic acid were designed as 40.812mM and 16mMrespectively. The acetylation level of histone H3K9 was detected by Western blot before and after CP15-13 intervention after low and high concentration of CP15-13. Fluorescence quantitative polymerase chain reaction (quantitative polymerase chain reaction, Q-PCR) based on SYBR GREEN 鈪，
本文编号：2203363
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