【作者】 钟立霖；

【导师】 田杰；

【作者基本信息】 重庆医科大学 ， 儿科学， 2010， 硕士

【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(P>0.05),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(P<0.05),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(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(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: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。更多还原

【Abstract】 BackgroundAlcohol exposure during pregnancy would cause congenital heart disease. However, the mechanisms responsible for it remain elusive. Recent evidence suggests that ethanol and its metabolites can selectively increase the acetylation of histone H3 at lysine 9(H3K9) in rat hepatocytes, which can be considered as a mechanism that alters gene expression. Heart development requires the sequential accurate expression of heart development-related genes such as GATA4,Mef2c,Tbx5 at different time and space. Inappropriate expression or repression of these genes can change trajectories and result in developmental malformation.ObjectiveThis study investigated the effect of ethanol and its metabolites on acetylation of H3K9 and the mRNA expression of these genes in cardiac progenitor cells 15-13(CP15-13). To confirm whether they can cause H3K9 epimutation. Materials and MethodsWe applied CP15-13 as our study object.After recovery from liquid nitrogen,the cells were cultured with high glucose DMEM medium containing 10% fetal bovine serum. At 80% confluence,the cells were subcultured for 3 passages.The fourth generation of cells are used in our experiments.We used MTT assay to investigate the cytotoxicity of ethanol and its metabolites to cardiac progenitor cell and select the low and high concentration intervention groups.After intervention with low and high concentration groups .We used Western-blot to detect acetylation of histone H3K9 and Q-PCR to detect the mRNA expression of heart development-related genes Gata4, Mef2c ,Tbx5 in cardiac progenitor cells.Results1.MTT assay showed that 50mM ethanol(0.368±0.028), 4mM acetaldehyd(e0.336±0.040), 4mM acetat(e0.359±0.014),as compared with contro(l0.358±0.066), didn’t affect the proliferation of cells (P>0.05).We used these concentrations as our low concentration groups. 200mM ethanol(0.245±0.042), 12mM acetaldehyde(0.220±0.017), 16mM acetate(0.243±0.024), as compared with control(0.358±0.066), inhibited the proliferation about 30%(P<0.05). We used these concentrations as our high concentration groups.2. After intervention with low and high concentration groups .We used Western-blot and Q-PCR respectively to detect the acetylation of histone H3K9 and the mRNA expression of heart development-related genes. We revealed that low concentration groups of 50mM ethanol, 4mM acetate respectively increased 2.4, 2.2-fold acetylation of H3K9(P<0.05),but the heart development-related genes had no significant change(P>0.05). High concentration groups of 200mM ethanol, 16mM acetate respectively increased 5.3,5.6-fold acetylation of H3K9(P<0.05)and the expression of heart development-related genes GATA4 increased respectively (1.767±0.173)、(1.518±0.133) , Mef2c increased respectively (3.301±0.465)、(1.875±0.587) , as compared with control or the corresponding low concentration group(sP<0.05). Whether 4mM or 12mM acetaldehyde all had a negligible effect on the acetylation of H3 lys9 and the expression of heart development-related genes(P>0.05).ConclusionsHigh concentrations of ethanol and its metabolites all have a cytotoxicity effect to cardiac progenitor cells. Ethanol and one of its metabolites acetate have the H3K9 epimutation effect,but its another metabolite acetaldehyde has no this effect.更多还原