【摘要】： 目的: 孕期酒精暴露可導(dǎo)致先天性心臟病(Congenital heart disease,CHD,簡(jiǎn)稱(chēng)先心病),但其具體機(jī)制目前仍不清楚。我們前期研究提示組蛋白乙�；揎検Ш饪梢鹦呐K發(fā)育相關(guān)基因表達(dá)異常(即表突變,Epimutation),可能與先心病發(fā)生有關(guān)。目前研究表明,乙醇可選擇性引起體外培養(yǎng)肝細(xì)胞組蛋白H3第9位賴(lài)氨酸(Histone H3 lysine 9,H3K9)乙�；揎検Ш�,但是否引起心臟中的H3K9乙酰化修飾失衡以及進(jìn)一步影響心臟發(fā)育相關(guān)基因的表達(dá)(即H3K9表突變)仍不清楚。本研究以心肌祖細(xì)胞15-13(Cardiac progenitor cells15-13,CP15-13)為研究對(duì)象,探討乙醇及其代謝產(chǎn)物對(duì)心肌祖細(xì)胞的毒性及H3K9表突變作用。 材料與方法: 以CP15-13為研究對(duì)象,復(fù)蘇細(xì)胞后,用含10%胎牛血清的DMEM高糖培養(yǎng)基培養(yǎng),待細(xì)胞貼滿(mǎn)瓶底80%左右時(shí)以1:3比例傳代,取第四代細(xì)胞做干預(yù)實(shí)驗(yàn)。應(yīng)用MTT比色法觀察乙醇及其代謝產(chǎn)物對(duì)心肌祖細(xì)胞的毒性作用及篩選出低、高濃度干預(yù)組。劑量設(shè)計(jì)為乙醇50,100,200mM,乙醛及乙酸均設(shè)計(jì)為4,8,12,16mM。低、高濃度干預(yù)CP15-13后,應(yīng)用Western blot方法檢測(cè)CP15-13干預(yù)前后組蛋白H3K9乙�；礁淖儭�(yīng)用基于SYBR GREENⅠ的熒光定量聚合酶鏈反應(yīng)(quantitative polymerase chain reaction, Q-PCR)方法檢測(cè)CP15-13心臟發(fā)育相關(guān)基因GATA4、Mef2c、Tbx5 mRNA表達(dá)量在干預(yù)前后的變化。 結(jié)果: 1.MTT結(jié)果顯示50 mM乙醇(0.368±0.028)、4 mM乙醛(0.336±0.040)、4 mM乙酸(0.359±0.014)與對(duì)照組(0.358±0.066)比較不影響心肌祖細(xì)胞增殖(P0.05),作為低濃度干預(yù)組, 200 mM乙醇(0.245±0.042)、12 mM乙醛(0.220±0.017)、16 mM乙酸(0.243±0.024)與對(duì)照組(0.358±0.066)比較,對(duì)心肌祖細(xì)胞抑制率為30%左右(P0.05),作為高濃度干預(yù)組。 2.MTT篩選出低、高濃度干預(yù)CP15-13后,應(yīng)用Western blot法及Q-PCR法分別對(duì)組蛋白H3K9乙�；郊靶呐K發(fā)育相關(guān)基因mRNA表達(dá)水平進(jìn)行對(duì)比,結(jié)果顯示低濃度組乙醇、乙酸分別使組蛋白H3K9乙�；缴�2.4、2.2倍(P0.05),心臟發(fā)育相關(guān)基因表達(dá)無(wú)明顯變化(P0.05),高濃度組乙醇、乙酸分別使組蛋白H3K9乙�；缴�5.3、5.6倍,同時(shí)心臟發(fā)育相關(guān)基因GATA4表達(dá)分別增加(1.767±0.173)、(1.518±0.133),Mef2c表達(dá)分別增加(3.301±0.465)、(1.875±0.587),與對(duì)照組及相應(yīng)低濃度組比較均有統(tǒng)計(jì)學(xué)差異(P0.05),乙醛則無(wú)論低濃度還是高濃度對(duì)組蛋白H3K9乙�；郊盎虮磉_(dá)均無(wú)明顯影響(P0.05)。 結(jié)論: 1.高濃度的乙醇及其代謝產(chǎn)物對(duì)心肌祖細(xì)胞均有毒性作用 2.乙醇及其代謝產(chǎn)物之一乙酸對(duì)心肌祖細(xì)胞具有組蛋白H3K9表突變作用,而其另一代謝產(chǎn)物乙醛無(wú)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，

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