BMP信号通路介导酒精致H9c2心肌细胞组蛋白高乙酰化上调心脏核心转录因子表达的研究

发布时间：2018-04-17 06:00

  本文选题：H9c2心肌细胞 + BMP信号通路　； 参考：《重庆医科大学》2014年硕士论文

【摘要】：目的： 采用Bone morphogenetic protein（骨形态形成蛋白，BMP）信号通路的特异性抑制剂dorsomorphin（DM）阻断其传导，以证实BMP信号通路介导酒精致组蛋白高乙酰化上调心脏核心转录因子表达的科学假说。 方法： （1）不同浓度酒精浓度（0mM、10mM、50mM、100mM、200mM、500mM和1000mM）处理H9c2细胞，36h后采用MTT检测各干预组H9c2细胞存活率，筛选最适酒精干预浓度。 （2）酒精干预细胞36h后采用Real-Time qRT-PCR方法检测BMPs各亚型mRNA的表达水平；加入不同DM浓度（0μM、1μM、2.5μM、5μM、10μM和20μM）阻断酒精作用，36h后采用Real-TimeqRT-PCR方法检测BMPs通路下游靶基因GATA4mRNA表达水平，，筛查出最适DM浓度。 （3）酒精和/或DM处理细胞36h后，应用Real-Time qRT-PCR技术检测心脏核心转录因子MEF2C、GATA4、Nkx2.5和Tbx5的表达，及Smad4mRNA的表达水平；采用Western-blotting技术检测H9c2细胞组蛋白H3总乙酰化水平和心脏发育相关基因Cx43表达水平；ChIP-Real-Time qPCR技术检测核心转录因子启动子区域组蛋白H3乙酰化水平。 结果： （1）10mM、50mM、100mM酒精干预36h后H9c2细胞生存率与对照组比较没有变化（P＞0.05），200mM、500mM和1000mM酒精使细胞生存率降低25.4%、37.0%和75.0%（P＜0.05）。 （2）100mM酒精干预36h后，H9c2心肌细胞BMP2（1.60±0.14）、BMP4（1.27±0.08）、BMP6（1.44±0.25）、BMP7（1.59±0.06）mRNA相对表达量均较对照组升高（P＜0.05）；BMP5（1.14±0.08）、BMP10（1.05±0.09）mRNA相对表达量较对照组有升高趋势，但差异无统计学意义（P＞0.05）。 （3）100mM酒精和不同浓度DM干预后，GATA4mRNA的表达呈现出先降低后上升的趋势，且在DM浓度为5μM时达到最低。 （4）100mM酒精可引起MEF2C（1.42±0.27）、GATA4（1.50±0.15）、Nkx2.5（1.41±0.18）和Smad4（1.78±0.15） mRNA表达水平升高（P0.05）；加入5μM DM后能降至正常水平（P0.05）；5μMDM可使MEF2C mRNA表达水平上升1.2倍（P0.05）；100mM酒精使Cx43蛋白表达水平升高2.7倍（P0.05）；加入5μM DM后能降至正常水平（P0.05）；100mM酒精、5μM DM对TBX5mRNA表达（0.98±0.08）、（0.95±0.23）无明显影响（P0.05）。 （5）100mM酒精可使组蛋白H3总乙酰化水平上升2.4倍（P0.05），加入5μM DM后降至对照组水平（P0.05）。 （6）100mM酒精能使MEF2C（1.56±0.24）、GATA4（2.04±0.69）和Nkx2.5（1.50±0.17）启动子区域的组蛋白H3乙酰化水平升高（P0.05），加入5μM DM后MEF2C和GATA4启动子区域乙酰化水平下降至正常（P0.05），Nkx2.5启动子区域乙酰化水平下调（1.25±0.16），但未降至正常（P0.05）。100mM酒精、5μMDM未对TBX5启动子区域组蛋白H3乙酰化（0.81±0.35）（、1.09±0.36）产生明显影响（P0.05）。 结论 （1）酒精可以引起与心脏相关的BMP亚型mRNA表达上升。 （2）BMP信号通路介导酒精对MEF2C、GATA4和Nkx2.5启动子区组蛋白H3乙酰化的促进作用可能是酒精引起MEF2C、 GATA4和Nkx2.5表达上调的机制之一。
[Abstract]:Objective:The transduction of Bone morphogenetic protein (BMP) was blocked by dorsomorphin DM1, a specific inhibitor of bone morphogenetic protein (BMP) signaling pathway, in order to confirm the scientific hypothesis that high acetylation of wine refined histone mediated by BMP signaling pathway upregulated the expression of cardiac core transcription factors.Methods:(1) H9c2 cells were treated with different concentrations of alcohol (0 mMU, 10 mMU, 50 mMU, 100 mMU, 500 mm and 1 000 mm) for 36 h, then the survival rate of H9c2 cells in each intervention group was determined by MTT, and the optimal concentration of alcohol intervention was screened.(2) the expression of mRNA in BMPs subtypes was detected by Real-Time qRT-PCR method after 36 h of alcohol intervention, and 10 渭 M and 20 渭 M of 5 渭 M ~ 5 渭 M ~ + 5 渭 M ~ + 5 渭 M ~ (5 渭 M) of different DM concentration were added to detect the GATA4mRNA expression in the downstream target gene of BMPs pathway by Real-TimeqRT-PCR method for 36 h, and the optimal DM concentration was screened out.(3) after alcohol and / or DM were treated for 36 h, the expression of MEF2CfGATA4Nkx2.5 and Tbx5 and the expression of Smad4mRNA were detected by Real-Time qRT-PCR technique.The total acetylation level of histone H3 in H9c2 cells and the expression level of cardiac development-related gene Cx43 were detected by Western-blotting and ChIP-Real-Time qPCR techniques were used to detect the acetylation level of histone H3 in core transcription factor promoter region.Results:Compared with the control group, the survival rate of H9c2 cells was not changed after alcohol treatment (P > 0.05). The survival rate of H9c2 cells decreased by 25.4mg / 37.0% and 75.0% (P < 0.05), compared with that of the control group (P < 0.05). The survival rate of H9c2 cells was significantly lower than that of the control group (P < 0.05). The survival rate of H9c2 cells was significantly lower than that of the control group (P > 0.05).The relative expression of BMP71.59 卤0.06)mRNA in H9c2 cardiomyocytes was significantly higher than that in the control group (1.44 卤0.25mM, 1.44 卤0.25mm). The relative expression of BMP51.14 卤0.08BMP100.05 卤0.09)mRNA in H9c2 cardiomyocytes was significantly higher than that in the control group (P < 0.05), but there was no significant difference between the two groups (P > 0.05).The expression of GATA4 mRNA decreased at first and then increased after the intervention of 100 mm alcohol and different concentrations of DM, and reached the lowest level when DM concentration was 5 渭 M.1. 50 卤0. 15 mRNA and 1. 41 卤0. 18) and Smad4(1.78 卤0. 15) increased mRNA expression level; 5 渭 M DM decreased MEF2C mRNA expression to normal level P0. 05 渭 MDM increased 1. 2 times P0. 05 mm alcohol increased Cx43 protein expression by 2. 7 times P0. 05%; 5 渭 M DM added 5 渭 M DM decreased to normal level P0. 05%, P0. 05%, P0. 05%, P0. 05%, P0. 05%, P0. 05%, P0. 05%, P0. 05%, P0. 05% and 5 渭 M DM. 5 渭 M DM added 5 渭 M DM decreased to normal level P0. 05% P0. 05% after adding 5 渭 M DM, the expression level of Cx43 protein decreased to normal level.There was no significant effect of 5 渭 M DM on the expression of TBX5mRNA 0.98 卤0.08 (0.95 卤0.23).The total acetylation level of histone H3 was increased by 2.4 times and decreased to the control level after adding 5 渭 M DM.The histone H3 acetylation level in MEF2C(1.56 卤0.24 Gata 42.04 卤0.69) and Nkx2.5(1.50 卤0.17) promoter region was increased by 100mm alcohol. The acetylation level of MEF2C and GATA4 promoter region decreased to 1.25 卤0.16m after adding 5 渭 M DM, but did not decrease to normal P0.05Nkx2.5 promoter region.Alcohol (5 渭 MDM) had no significant effect on the histone H3 acetylation of TBX5 promoter (0.81 卤0.35) (1.09 卤0.36).ConclusionAlcohol can increase the expression of BMP subtype mRNA associated with the heart.The effect of alcohol on the acetylation of histone H3 in MEF2CnGATA4 and Nkx2.5 promoter may be one of the mechanisms of the up-regulation of MEF2C, GATA4 and Nkx2.5 induced by alcohol.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R725.4

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