【作者】 郑敏；

【导师】 田杰；

【作者基本信息】 重庆医科大学 ， 儿科学， 2013， 博士

【摘要】 目的构建BMP2过表达的心肌细胞模型，检测BMP2对心脏核心转录因子GATA4、MEF2C和Tbx5表达的影响，并研究BMP2对H9c2心肌细胞总组蛋白H3、基因启动子区组蛋白H3乙酰化修饰及组蛋白乙酰化酶（HATs）亚型p300和GCN5的调控作用，以证实我们的科学假设：BMP2是心肌细胞组蛋白乙酰化修饰的上游信号通路之一。方法（1）过表达BMP2的腺病毒（AdBMP2）及对照空腺病毒（AdGFP）在HEK293细胞中扩增后，分别以不同滴度转染大鼠H9c2心肌细胞，24h后荧光倒置显微镜下观察AdBMP2/AdGFP腺病毒转染细胞绿色荧光蛋白的表达情况，流式细胞术检测AdBMP2/AdGFP腺病毒转染效率。（2）AdBMP2/AdGFP腺病毒转染H9c2心肌细胞24h、48h和72h后收集细胞，提取mRNA，Real-Time qRT-PCR检测各处理组细胞BMP2、MEF2C、GATA4和Tbx5及HATs亚型p300和GCN5的mRNA表达水平，筛选最佳干预时间。（3）AdBMP2/AdGFP腺病毒转染H9c2心肌细胞48h后收集细胞，比色法检测各处理组H9c2心肌细胞核蛋白HATs活性，Western-blotting检测各处理组细胞总组蛋白H3的乙酰化水平，ChIP-Real-Time qPCR检测各处理组细胞MEF2C、GATA4和Tbx5启动子区组蛋白H3乙酰化水平。结果（1）AdBMP2转染H9c2心肌细胞24h后，荧光倒置显微镜下可见细胞中出现大量绿色荧光，流式细胞术检测结果显示AdBMP2转染效率可达90%以上。（2）AdBMP2转染H9c2心肌细胞24h、48h、72h后，BMP2及心脏核心转录因子MEF2C和GATA4的mRNA表达水平较对照组明显升高（P<0.05），并于转染后48h达到高峰，而Tbx5的表达没有明显变化。（3）AdBMP2转染H9c2心肌细胞48h后，HATs亚型p300的mRNA表达水平较对照组升高（P<0.05），而GCN5的表达水平与对照组相比没有明显的变化。（4）AdBMP2转染H9c2心肌细胞48h后，细胞核蛋白HATs活性较对照组明显升高（P<0.05），总组蛋白H3乙酰化水平也较对照组明显上调（P<0.05），MEF2C、GATA4启动子区组蛋白H3乙酰化水平亦相应升高（P<0.05），但Tbx5启动子区组蛋白H3乙酰化水平与对照组相比未见明显变化。结论（1）BMP2可上调H9c2心肌细胞中心脏核心转录因子MEF2C和GATA4的表达，而对Tbx5的表达没有影响。（2）BMP2可引起H9c2心肌细胞组蛋白H3高乙酰化，可能是H9c2心肌细胞组蛋白乙酰化修饰的上游信号通路之一。（3）BMP2引起的H9c2心肌细胞组蛋白H3高乙酰化可能与HATs亚型p300有关。（4）BMP2对MEF2C和GATA4启动子区组蛋白H3乙酰化的促进作用可能是BMP2引起MEF2C和GATA4表达上调的机制之一，而Tbx5启动子区组蛋白H3乙酰化未受BMP2的影响可能是Tbx5的表达不受BMP2调控的原因之一。目的使用HATs亚型p300的抑制剂姜黄素（Curcumin）来研究p300在BMP2致H9c2心肌细胞心脏核心转录因子GATA4和MEF2C高表达及组蛋白高乙酰化中的作用，以证实我们的科学假设：p300参与BMP2对H9c2心肌细胞心脏核心转录因子表达及组蛋白乙酰化修饰的调控作用。方法（1）AdBMP2转染H9c2心肌细胞后，用不同浓度的p300HAT活性抑制剂姜黄素(10μM,20μM,30μM,40μM)处理细胞(6h,12h,24h,48h)，比色法检测各处理组细胞HATs活性，筛选姜黄素最佳处理浓度及时间。（2）AdBMP2和/或姜黄素处理H9c2心肌细胞后，Real-Time qRT-PCR检测各处理组细胞心脏核心转录因子GATA4、MEF2C和Tbx5及HATs亚型p300和GCN5的表达水平，Western-blotting检测各处理组细胞总组蛋白H3的乙酰化水平，ChIP-Real-Time qPCR检测各处理组细胞GATA4、MEF2C和Tbx5启动子区组蛋白H3乙酰化水平。结果（1）不同浓度（10μM,20μM,30μM,40μM)的姜黄素处理细胞24h后，H9c2心肌细胞的HATs活性明显下降（P<0.05），并于40μM达到最低点。40μM姜黄素分别处理细胞6h,12h,24h,48h后，HATs活性于处理后12h达到最低点。（2）AdBMP2和姜黄素共同处理H9c2心肌细胞后，心脏核心转录因子GATA4和MEF2C及HATs亚型p300的mRNA表达水平较单独AdBMP2处理组明显下降（P<0.05），而Tbx5及GCN5的mRNA表达水平在各处理组之间没有明显的变化。（3）AdBMP2与姜黄素共同处理H9c2心肌细胞后，细胞中总组蛋白H3乙酰化水平及GATA4和MEF2C启动子区组蛋白H3乙酰化水平较单独AdBMP2处理组明显下降（P<0.05），而Tbx5启动子区组蛋白H3乙酰化在各处理组之间没有明显的变化。结论（1）姜黄素可抑制HATs亚型p300的表达，而对GCN5的表达没有影响。（2）在H9c2心肌细胞中，p300HAT活性抑制剂姜黄素可拮抗BMP2引起的组蛋白H3高乙酰化及GATA4和MEF2C的高表达，说明p300参与BMP2对H9c2心肌细胞GATA4和MEF2C表达及组蛋白乙酰化的调控。（3）在H9c2心肌细胞中，Tbx5启动子区组蛋白H3乙酰化及其表达可能不受p300的调控。目的使用BMPs信号通路抑制剂dorsomorphin（DM）来研究BMPs在氧化应激反应所致H9c2心肌细胞组蛋白高乙酰化中的作用，以证实我们的科学假设：BMPs参与介导氧化应激反应所致H9c2心肌细胞组蛋白高乙酰化。方法（1）不同浓度H2O2(50μM、100μM、150μM、200μM、250μM、300μM、350μM、400μM)处理H9c2心肌细胞，24h后采用MTT法检测各处理组细胞的存活率。（2）5μM的BMPs信号通路抑制剂DM和/或适宜浓度的H2O2处理细胞，Real-Time qRT-PCR检测各处理组细胞BMP2及心脏核心转录因子GATA4，MEF2C和Tbx5的表达水平，Western-blotting检测各处理组细胞总组蛋白H3的乙酰化水平。结果（1）50、100和150μM浓度的H2O2对H9c2心肌细胞的存活率没有明显的影响，200、250、300、350、400和450μM浓度的H2O2处理H9c2心肌细胞后，细胞的存活率分别降低10.5％、16.9％、21.9％、32.4％、47.0％和58.6％。（2）400μM H2O2处理H9c2心肌细胞后，BMP2、GATA4、MEF2C和Tbx5的mRNA表达水平较空白对照组明显升高（P<0.05），细胞组蛋白H3的乙酰化水平亦相应升高（P<0.05）。（3）400μM H2O2和DM共同处理H9c2心肌细胞后，H9c2心肌细胞中总组蛋白H3乙酰化水平较单独400μM H2O2处理组有所下降（P<0.05），GATA4和Tbx5的mRNA表达水平也较单独400μMH2O2处理组有所降低（P<0.05），而MEF2C的mRNA表达水平较单独400μM H2O2处理组有所升高（P<0.05）。结论（1）利用H2O2成功构建H9c2心肌细胞氧化损伤模型。（2）400μM H2O2引起的氧化应激可上调H9c2心肌细胞BMP2、GATA4、MEF2C和Tbx5的表达并引起细胞组蛋白H3高乙酰化。（3）BMPs信号通路抑制剂DM对400μM H2O2引起的氧化应激所致H9c2心肌细胞组蛋白H3高乙酰化及GATA4和Tbx5表达上调具有一定的拮抗作用，说明BMPs（BMP2及其它BMPs亚型）参与介导氧化应激引起的心肌细胞组蛋白高乙酰化及GATA4和Tbx5的高表达，提示在氧化应激的病理状态下，BMPs可能也是心肌细胞组蛋白乙酰化修饰上游信号通路的组成部分，也提示氧化应激可能激活了上调Tbx5表达的BMPs亚型。（4）BMPs信号通路抑制剂DM可引起H9c2心肌细胞MEF2C的表达升高，提示在H9c2心肌细胞中，BMPs各亚型的综合效应可能是对MEF2C的表达起抑制作用，也提示氧化应激可能通过其它信号通路调控MEF2C的表达。更多还原

【Abstract】 ObjectiveBuilding the myocardial cell model that overexpressing BMP2to studythe effects of BMP2on the expression of cardiac core transcription factorsGATA4, MEF2C and Tbx5, the histone acetylation level both in the wholechromatin and in the promoter regions of GATA4, MEF2C and Tbx5, andthe expression of histone deacetylase (HAT) p300and GCN5. In the presentstudy, we will test the hypothesis that BMP2acts as one of the upstreamregulators of histone acetylation in cardiomyocytes.Methods（1）Rat H9c2myocardial cells(H9c2cells) were transfected withAdBMP2or AdGFP adenovirus that amplified in HEK293cells, theexpression of green fluorescent protein(GFP) were detected under aninverted fluorescence microscope, and the transfection efficiency weredetected by flow cytometry24h after transfection. （2）24h,48h and72h after transfected with AdBMP2, H9c2cells werecollected to extract mRNA, Real-Time qRT-PCR were used to detectthe mRNA expression levels of BMP2, MEF2C, GATA4, Tbx5, p300and GCN5.（3）48h after transfected with AdBMP2, H9c2cells were collected,colorimetric assay were used to detect HAT activity, Western-blottingwere used to detect the histone H3acetylation levels in the wholechromatin. ChIP-Real-Time qPCR were used to detect the histone H3acetylation level in the promoter regions of GATA4, MEF2C andTbx5.Results（1）24h after AdBMP2transfection, high expression level of GFP werevisible in the cells under the inverted fluorescence microscope. Theresults of flow cytometry showed that the transfection efficiency wasup to90%.（2）24h,48h,72h after AdBMP2transfection, the mRNA expression levelof BMP2and the cardiac core transcription factor MEF2C andGATA4were significantly increased compared with the controlcells(P<0.05) and reached the peak at48h after transfection inH9c2cells. However the expression of Tbx5was not affected in thesame cells after AdBMP2transfection.（3）48h after AdBMP2transfection, the mRNA expression level of HAT p300was upregulated compared with the control cells(P<0.05), whilethe expression of another HAT GCN5was not changed.（4）48h after AdBMP2transfection, HAT activity and the histone H3acetylation level in the whole chromatin were significantly increasedcompared with the control H9c2cells(P<0.05). In addition, thehistone H3acetylation level in the promoter regions of GATA4andMEF2C were also increased compared with the control cells(P<0.05).However the histone H3acetylation level in the promoter region ofTbx5was not changed.Conclusions（1）BMP2upregulates the expression of cardiac core transcription factorsMEF2C and GATA4, but not Tbx5.（2）BMP2-induced histone H3hyperacetylation in H9c2cells revealedthat BMP2acts as one of the upstream regulators of histoneacetylation in cardiomyocytes.（3）BMP2-induced histone H3hyperacetylation in H9c2cells maybeassociated with the HAT p300.（4）Histone H3acetylation may be one of the molecular mechanisms bywhich BMP2upregulates the expression of GATA4and MEF2C.However, the unaffected expression of Tbx5maybe associated withthe unchanged histone H3acetylation in the promoter region of Tbx5in H9c2cells overexpressing BMP2. ObjectiveHAT p300inhibitor curcumin was used to research the effect of p300on the BMP2-induced histone hyperacetylation and the BMP2-inducedupregulation of cardiac core transcription factors GATA4and MEF2C, totest the hypothesis that p300is involved in the regulation of cardiac coretranscription factors and the histone H3acetylation by BMP2in H9c2cells.Methods（1）The AdBMP2transfected H9c2cells were treated with differentconcentrations(10μM,20μM,30μM,40μM) of the HAT p300inhibitorcurcumin for various treatment times (6h,12h,24h,48h), colorimetricassay were used to detect HAT activity and to find an optimumconcentration and time duration.（2）After AdBMP2and/or curcumin treatment, H9c2cells were collected.Real-Time qRT-PCR were used to detect the expression levels of cardiac core transcription factor GATA4, MEF2C and Tbx5,Western-blotting were used to detect histone H3acetylation level inthe whole chromatin, and ChIP-Real-Time qPCR were used to detectthe histone H3acetylation levels in the promoter regions of GATA4,MEF2C and Tbx5.Results（1）24h after treated with different concentrations (10μM,20μM,30μM,40μM) of curcumin, HAT activity was decreased(P<0.05) and reachedits lowest point at40μM in AdBMP2transfected H9c2cells. Aftertreated with40μM curcumin for various treatment times (6h,12h,24h,48h), HAT activity was decreased(P<0.05) and reached its lowestpoint at12h after treatment in AdBMP2transfected H9c2cells.（2）The mRNA expression levels of cardiac core transcription factorsGATA4and MEF2C and HAT p300were decreased(P<0.05) inAdBMP2transfected and curcumin treated cells compared with theAdBMP2transfection group, while the mRNA expression levels ofTbx5and GCN5were not changed.（3）The histone H3acetylation level in the whole chromatin and in thepromoter regions of GATA4and MEF2C were also decreased(P<0.05)in AdBMP2transfected and curcumin treated cells compared with theAdBMP2transfection group, while the histone H3acetylation level inthe promoter region of Tbx5was not changed. Conclusions（1）HAT p300inhibitor curcumin is able to inhibit the expression of p300but not GCN5.（2）BMP2-induced histone hyperacetylation and the upregulation ofcardiac core transcription factors GATA4and MEF2C are antagonizedby curcumin, suggesting that p300is involved in the regulation ofcardiac core transcription factors and the histone H3acetylation byBMP2.（3）The expression and the histone H3acetylation in the promoter regionof Tbx5maybe not regulated by p300in H9c2cells. ObjectiveBMPs signal inhibitor dorsomorphin (DM) was used to research theeffect of BMPs signal on the oxidative stress-induced histonehyperacetylation, to test the hypothesis that BMPs are involved in theoxidative stress-induced histone hyperacetylation in H9c2cells Methods（1）H9c2cells were treated with different concentrations of H2O2(50μM,100μM,150μM,200μM,250μM,300μM,350μM,400μM) for24h,MTT assay were used to detect cell survival rate, and to select theappropriate concentration of H2O2to build the oxidative injury modelof H9c2cells.（2）H9c2cell were treated with5μM BMPs signal inhibitor dorsomorphin(DM) and/or the appropriate concentration of H2O2. Real-TimeqRT-PCR were used to detect the mRNA expression levels of BMP2and cardiac core transcription factors GATA4, MEF2C and Tbx5.Western-blotting assay were used to detect histone H3acetylationlevel in the whole chromatin.Results（1）The H9c2cell survival rates were not changed after50,100and150μM H2O2treatment, and after treatment with200,250,300,350,400and450μM H2O2, the cell survival rate were reduced by10.5%,16.9%,21.9%,32.4%,47.0%and58.6%respectively.（2）In400μM H2O2treated H9c2cells, The histone H3acetylation levelin the whole chromatin and the mRNA expression levels of BMP2,GATA4, MEF2C and Tbx5were significantly increased(P<0.05)compared with blank control cells.（3） The histone H3acetylation level in the whole chromatin and themRNA expression levels of GATA4and Tbx5were decreased(P<0.05) in400μM H2O2and DM treated H9c2cells compared with400μMH2O2treated group. However, the mRNA expression level of MEF2Cwere increased(P<0.05) in DM and400μM H2O2treated H9c2cellscompared with400μM H2O2treated group.Conclusions（1）The oxidative injury model of H9c2cells were successfullyconstructed with H2O2.（2）The expression of BMP2, GATA4, MEF2C and Tbx5are upregulatedby400μM H2O2-induced oxidative stress.（3）400μM H2O2-induced histone H3acetylation and the expression ofGATA4and Tbx5is partially antagonized by BMPs signal inhibitorDM in H9c2cells, suggesting that BMPs(BMP2and other BMPssubtypes) is involved in oxidative stress-induced histone acetylationand the expression of GATA4and Tbx5, indicating that in thepathological state(oxidative stress), BMPs may also act as theupstream signal pathways of histone acetylation in cardiomyocytes,and also indicating that certain BMPs subtypes that upregulate theexpression of Tbx5maybe induced under oxidative stress.（4）The expression of MEF2C is enhanced by BMPs signal inhibitor DMin H9c2cells, suggesting that the expression of MEF2C maybeinhibited by the combined effects of BMPs subtypes, and alsoindicating that other signals maybe in involved in oxidative stress-induced expression of MEF2C.更多还原