BMP2对H9c2心肌细胞组蛋白乙酰化修饰的调控作用及其机制研究
发布时间：2018-07-31 07:07
【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（P0.05），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（P0.05），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（P0.05），总组蛋白H3乙酰化水平也较对照组明显上调（P0.05），MEF2C、GATA4启动子区组蛋白H3乙酰化水平亦相应升高（P0.05），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（P0.05），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（P0.05），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（P0.05），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（P0.05），细胞组蛋白H3的乙酰化水平亦相应升高（P0.05）。 （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（P0.05），GATA4和Tbx5的mRNA表达水平也较单独400μMH2O2处理组有所降低（P0.05），而MEF2C的mRNA表达水平较单独400μM H2O2处理组有所升高（P0.05）。 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。
[Abstract]:objective
The effects of BMP2 on the expression of core transcription factors GATA4, MEF2C and Tbx5 were constructed by constructing BMP2 overexpressed cardiomyocytes, and the regulation of BMP2 on the total histone H3 of H9c2 cardiomyocytes, H3 acetylation of the promoter region histone and the P300 of histone acetyltransferase (HATs) subtype and GCN5 were studied. BMP2 is one of the upstream signaling pathways of histone acetylation in cardiomyocytes.
Method
(1) the adenovirus (AdBMP2) expressing BMP2 and the control space adenovirus (AdGFP) were amplified in HEK293 cells and transfected to H9c2 cardiomyocytes at different titers. The expression of green fluorescent protein (GFP) of AdBMP2/AdGFP adenovirus transfected cells was observed after 24h fluorescence inversion microscope. The transfection efficiency of AdBMP2/AdGFP adenovirus was detected by flow cytometry.
(2) AdBMP2/AdGFP adenovirus transfected H9c2 cardiomyocytes 24h, 48h and 72h to collect cells and extract mRNA. Real-Time qRT-PCR was used to detect the expression level of BMP2, MEF2C, GATA4, Tbx5 and subtypes, and to screen the best intervention time.
(3) AdBMP2/AdGFP adenovirus transfected into H9c2 cardiomyocytes and 48h cells were collected to collect cells, and the cell nuclear protein HATs activity of H9c2 myocardium in each treatment group was detected by colorimetric assay. Western-blotting was used to detect the level of acetylation of total histone H3 in each treatment group. ChIP-Real-Time qPCR detected MEF2C, GATA4 and Tbx5 promoter histone acetylated water in each processing group. Flat.
Result
(1) after transfection of 24h to H9c2 cardiomyocytes by AdBMP2, a large number of green fluorescence were found in the cells under the fluorescence inverted microscope. The results of flow cytometry showed that the transfection efficiency of AdBMP2 could reach more than 90%.
(2) the mRNA expression level of BMP2 and cardiac core transcription factor MEF2C and GATA4 increased significantly after AdBMP2 transfection of 24h, 48h, 72h, BMP2 and cardiac core transcription factor MEF2C and GATA4 (P0.05), and the 48h reached the peak after transfection, but the expression of Tbx5 was not significantly changed.
(3) after AdBMP2 transfected with H9c2 48h, the mRNA expression level of HATs subtype P300 was higher than that of the control group (P0.05), but the expression level of GCN5 was not significantly changed compared with the control group.
(4) after AdBMP2 transfection of H9c2 cardiomyocytes 48h, the activity of nuclear protein HATs was significantly higher than that of the control group (P0.05), and the level of total histone H3 acetylation was also significantly up (P0.05), MEF2C, and GATA4 promoter region histone H3 acetylation level was also increased (P0.05), but the level of protein acetylation in the Tbx5 promoter region was not compared with the control group. See obvious changes.
conclusion
(1) BMP2 could increase the expression of cardiac core transcription factors MEF2C and GATA4 in H9c2 cardiomyocytes, but had no effect on the expression of Tbx5.
(2) BMP2 can induce histone H3 hyperacetylation in H9c2 cardiomyocytes, which may be one of the upstream signaling pathways of histone acetylation modification in H9c2 cardiomyocytes.
(3) high acetylation of histone H3 induced by BMP2 may be related to HATs subtype P300 in H9c2.
(4) the promotion of BMP2 on the histone H3 acetylation of the promoter region of MEF2C and GATA4 may be one of the mechanisms of MEF2C and GATA4 up-regulated expression by BMP2, and the H3 acetylation of the Tbx5 promoter region histone is not affected by BMP2, which may be one of the reasons for Tbx5 expression not regulated by BMP2.
objective
HATs subtype P300 inhibitor curcumin (Curcumin) was used to study the role of P300 in BMP2 induced cardiac transcriptional factor GATA4 and MEF2C high expression and histone histone acetylation, in order to confirm our scientific hypothesis: P300 participates in the expression of cardiac nuclear transcription factors and histone acetylation modification in BMP2 for H9c2 cardiomyocytes. Regulation and control.
Method
(1) after transfection of AdBMP2 to H9c2 cardiomyocytes, the cells (6h, 12h, 24h, 48h) were treated with curcumin (10 M, 20 u M, 30, M, 40 M) with different concentrations of p300HAT activity inhibitor curcumin (6h, 12h, 24h, 48h). The cell HATs activity was detected by colorimetry, and the best treatment concentration and time of curcumin were screened.
(2) after AdBMP2 and / or curcumin treated H9c2 cardiomyocytes, Real-Time qRT-PCR was used to detect the expression level of GATA4, MEF2C, Tbx5 and HATs subtypes P300 and GCN5 in each treatment group. The level of acetylation of histone H3 in the promoter region of 2C and Tbx5.
Result
(1) after 24h of different concentrations (10 M, 20, M, 30 M, 40 M), the HATs activity of H9c2 cardiomyocytes decreased significantly (P0.05), and reached the lowest point at the lowest point of 40 micron.40 mu M curcumin respectively.
(2) after AdBMP2 and curcumin combined with H9c2 cardiomyocytes, the mRNA expression level of cardiac core transcription factor GATA4 and MEF2C and HATs subtype P300 was significantly lower than that of the single AdBMP2 treatment group (P0.05), while Tbx5 and GCN5 mRNA expression levels were not significantly altered between the treatment groups.
(3) after the combination of AdBMP2 and curcumin on H9c2 cardiomyocytes, the level of total histone H3 acetylation and the level of H3 acetylation in GATA4 and MEF2C promoter region were significantly lower than those in the single AdBMP2 treatment group (P0.05), but there was no significant change between the Tbx5 promoter region histone H3 acetylation in each treatment group.
conclusion
(1) curcumin inhibited the expression of P300 in HATs subtype but had no effect on the expression of GCN5.
(2) in H9c2 cardiomyocytes, p300HAT active inhibitor curcumin can antagonize the high acetylation of histone H3 and the high expression of GATA4 and MEF2C caused by BMP2, indicating that P300 participates in the regulation of BMP2 on GATA4 and MEF2C expression of H9c2 myocardial cells and histone acetylation.
(3) in H9c2 cardiomyocytes, acetylation and histone H3 expression in Tbx5 promoter region may not be regulated by p300.
objective
The use of BMPs signaling pathway inhibitor dorsomorphin (DM) to study the role of BMPs in the high acetylation of the H9c2 myocardial histone induced by oxidative stress to confirm our scientific hypothesis: BMPs is involved in the high acetylation of the protein in the H9c2 cardiomyocytes induced by oxidative stress reaction.
Method
(1) different concentrations of H2O2 (50, 50, 100, 150, 150, 200, 250, 250, 300, M, 350, and 400 micron) were treated for H9c2 cardiomyocytes, and the survival rate of each group was detected by MTT method after 24h.
(2) the BMPs signaling pathway inhibitor DM of 5 M and / or the appropriate concentration of H2O2 processing cells. Real-Time qRT-PCR was used to detect the BMP2 and cardiac transcriptional factor GATA4, MEF2C and Tbx5 expression levels in each treatment group, and to detect the level of acetylation of the total histone H3 in each treatment group.
Result
(1) the H2O2 concentration of 50100 and 150 mu M had no significant effect on the survival rate of H9c2 cardiomyocytes. The survival rate of cells decreased by 10.5%, 16.9%, 21.9%, 32.4%, 47% and 58.6%. respectively after 200250300350400 and 450 micron M H2O2 treatment of H9c2 cardiac myocytes.
(2) after 400 M H2O2 treatment of H9c2 cardiac myocytes, the level of mRNA expression in BMP2, GATA4, MEF2C and Tbx5 was significantly higher than that in the blank control group (P0.05), and the level of acetylation of the cell protein H3 was also increased (P0.05).
(3) after the combined treatment of H9c2 cardiomyocytes by 400 M H2O2 and DM, the level of total histone acetylation in H9c2 cardiac myocytes was lower than that in the 400 u M H2O2 treatment group (P0.05), and the GATA4 and Tbx5 mRNA expression levels were also lower than those of the single 400 mu treatment group. P0.05).
conclusion
(1) using H2O2 to successfully construct H9c2 myocardial cell oxidative damage model.
(2) Oxidative stress induced by 400 mu M H2O2 can up-regulate the expression of BMP2, GATA4, MEF2C and Tbx5 in H9c2 cardiomyocytes and induce the hyperacetylation of histone H3.
(3) BMPs signaling pathway inhibitor DM has a certain antagonistic effect on the high acetylation of the histone H3 and the up regulation of the expression of GATA4 and Tbx5 induced by oxidative stress caused by 400 M H2O2, indicating that BMPs (BMP2 and other BMPs subtypes) involved in the high acetylation of histones in cardiac myocytes and the high expression of GATA4 and proteins induced by oxidative stress. In the pathological state of oxidative stress, BMPs may also be part of the upstream signaling pathway in the acetylation of cardiac myocyte histone, suggesting that oxidative stress may activate the BMPs subtype of up regulation of Tbx5 expression.
(4) BMPs signaling pathway inhibitor DM can induce the increase of MEF2C expression in H9c2 cardiomyocytes, suggesting that the comprehensive effects of BMPs subtypes in H9c2 cardiomyocytes may inhibit the expression of MEF2C, suggesting that oxidative stress may regulate the expression of MEF2C through other signaling pathways.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R725.4
本文编号：2154819
[Abstract]:objective
The effects of BMP2 on the expression of core transcription factors GATA4, MEF2C and Tbx5 were constructed by constructing BMP2 overexpressed cardiomyocytes, and the regulation of BMP2 on the total histone H3 of H9c2 cardiomyocytes, H3 acetylation of the promoter region histone and the P300 of histone acetyltransferase (HATs) subtype and GCN5 were studied. BMP2 is one of the upstream signaling pathways of histone acetylation in cardiomyocytes.
Method
(1) the adenovirus (AdBMP2) expressing BMP2 and the control space adenovirus (AdGFP) were amplified in HEK293 cells and transfected to H9c2 cardiomyocytes at different titers. The expression of green fluorescent protein (GFP) of AdBMP2/AdGFP adenovirus transfected cells was observed after 24h fluorescence inversion microscope. The transfection efficiency of AdBMP2/AdGFP adenovirus was detected by flow cytometry.
(2) AdBMP2/AdGFP adenovirus transfected H9c2 cardiomyocytes 24h, 48h and 72h to collect cells and extract mRNA. Real-Time qRT-PCR was used to detect the expression level of BMP2, MEF2C, GATA4, Tbx5 and subtypes, and to screen the best intervention time.
(3) AdBMP2/AdGFP adenovirus transfected into H9c2 cardiomyocytes and 48h cells were collected to collect cells, and the cell nuclear protein HATs activity of H9c2 myocardium in each treatment group was detected by colorimetric assay. Western-blotting was used to detect the level of acetylation of total histone H3 in each treatment group. ChIP-Real-Time qPCR detected MEF2C, GATA4 and Tbx5 promoter histone acetylated water in each processing group. Flat.
Result
(1) after transfection of 24h to H9c2 cardiomyocytes by AdBMP2, a large number of green fluorescence were found in the cells under the fluorescence inverted microscope. The results of flow cytometry showed that the transfection efficiency of AdBMP2 could reach more than 90%.
(2) the mRNA expression level of BMP2 and cardiac core transcription factor MEF2C and GATA4 increased significantly after AdBMP2 transfection of 24h, 48h, 72h, BMP2 and cardiac core transcription factor MEF2C and GATA4 (P0.05), and the 48h reached the peak after transfection, but the expression of Tbx5 was not significantly changed.
(3) after AdBMP2 transfected with H9c2 48h, the mRNA expression level of HATs subtype P300 was higher than that of the control group (P0.05), but the expression level of GCN5 was not significantly changed compared with the control group.
(4) after AdBMP2 transfection of H9c2 cardiomyocytes 48h, the activity of nuclear protein HATs was significantly higher than that of the control group (P0.05), and the level of total histone H3 acetylation was also significantly up (P0.05), MEF2C, and GATA4 promoter region histone H3 acetylation level was also increased (P0.05), but the level of protein acetylation in the Tbx5 promoter region was not compared with the control group. See obvious changes.
conclusion
(1) BMP2 could increase the expression of cardiac core transcription factors MEF2C and GATA4 in H9c2 cardiomyocytes, but had no effect on the expression of Tbx5.
(2) BMP2 can induce histone H3 hyperacetylation in H9c2 cardiomyocytes, which may be one of the upstream signaling pathways of histone acetylation modification in H9c2 cardiomyocytes.
(3) high acetylation of histone H3 induced by BMP2 may be related to HATs subtype P300 in H9c2.
(4) the promotion of BMP2 on the histone H3 acetylation of the promoter region of MEF2C and GATA4 may be one of the mechanisms of MEF2C and GATA4 up-regulated expression by BMP2, and the H3 acetylation of the Tbx5 promoter region histone is not affected by BMP2, which may be one of the reasons for Tbx5 expression not regulated by BMP2.
objective
HATs subtype P300 inhibitor curcumin (Curcumin) was used to study the role of P300 in BMP2 induced cardiac transcriptional factor GATA4 and MEF2C high expression and histone histone acetylation, in order to confirm our scientific hypothesis: P300 participates in the expression of cardiac nuclear transcription factors and histone acetylation modification in BMP2 for H9c2 cardiomyocytes. Regulation and control.
Method
(1) after transfection of AdBMP2 to H9c2 cardiomyocytes, the cells (6h, 12h, 24h, 48h) were treated with curcumin (10 M, 20 u M, 30, M, 40 M) with different concentrations of p300HAT activity inhibitor curcumin (6h, 12h, 24h, 48h). The cell HATs activity was detected by colorimetry, and the best treatment concentration and time of curcumin were screened.
(2) after AdBMP2 and / or curcumin treated H9c2 cardiomyocytes, Real-Time qRT-PCR was used to detect the expression level of GATA4, MEF2C, Tbx5 and HATs subtypes P300 and GCN5 in each treatment group. The level of acetylation of histone H3 in the promoter region of 2C and Tbx5.
Result
(1) after 24h of different concentrations (10 M, 20, M, 30 M, 40 M), the HATs activity of H9c2 cardiomyocytes decreased significantly (P0.05), and reached the lowest point at the lowest point of 40 micron.40 mu M curcumin respectively.
(2) after AdBMP2 and curcumin combined with H9c2 cardiomyocytes, the mRNA expression level of cardiac core transcription factor GATA4 and MEF2C and HATs subtype P300 was significantly lower than that of the single AdBMP2 treatment group (P0.05), while Tbx5 and GCN5 mRNA expression levels were not significantly altered between the treatment groups.
(3) after the combination of AdBMP2 and curcumin on H9c2 cardiomyocytes, the level of total histone H3 acetylation and the level of H3 acetylation in GATA4 and MEF2C promoter region were significantly lower than those in the single AdBMP2 treatment group (P0.05), but there was no significant change between the Tbx5 promoter region histone H3 acetylation in each treatment group.
conclusion
(1) curcumin inhibited the expression of P300 in HATs subtype but had no effect on the expression of GCN5.
(2) in H9c2 cardiomyocytes, p300HAT active inhibitor curcumin can antagonize the high acetylation of histone H3 and the high expression of GATA4 and MEF2C caused by BMP2, indicating that P300 participates in the regulation of BMP2 on GATA4 and MEF2C expression of H9c2 myocardial cells and histone acetylation.
(3) in H9c2 cardiomyocytes, acetylation and histone H3 expression in Tbx5 promoter region may not be regulated by p300.
objective
The use of BMPs signaling pathway inhibitor dorsomorphin (DM) to study the role of BMPs in the high acetylation of the H9c2 myocardial histone induced by oxidative stress to confirm our scientific hypothesis: BMPs is involved in the high acetylation of the protein in the H9c2 cardiomyocytes induced by oxidative stress reaction.
Method
(1) different concentrations of H2O2 (50, 50, 100, 150, 150, 200, 250, 250, 300, M, 350, and 400 micron) were treated for H9c2 cardiomyocytes, and the survival rate of each group was detected by MTT method after 24h.
(2) the BMPs signaling pathway inhibitor DM of 5 M and / or the appropriate concentration of H2O2 processing cells. Real-Time qRT-PCR was used to detect the BMP2 and cardiac transcriptional factor GATA4, MEF2C and Tbx5 expression levels in each treatment group, and to detect the level of acetylation of the total histone H3 in each treatment group.
Result
(1) the H2O2 concentration of 50100 and 150 mu M had no significant effect on the survival rate of H9c2 cardiomyocytes. The survival rate of cells decreased by 10.5%, 16.9%, 21.9%, 32.4%, 47% and 58.6%. respectively after 200250300350400 and 450 micron M H2O2 treatment of H9c2 cardiac myocytes.
(2) after 400 M H2O2 treatment of H9c2 cardiac myocytes, the level of mRNA expression in BMP2, GATA4, MEF2C and Tbx5 was significantly higher than that in the blank control group (P0.05), and the level of acetylation of the cell protein H3 was also increased (P0.05).
(3) after the combined treatment of H9c2 cardiomyocytes by 400 M H2O2 and DM, the level of total histone acetylation in H9c2 cardiac myocytes was lower than that in the 400 u M H2O2 treatment group (P0.05), and the GATA4 and Tbx5 mRNA expression levels were also lower than those of the single 400 mu treatment group. P0.05).
conclusion
(1) using H2O2 to successfully construct H9c2 myocardial cell oxidative damage model.
(2) Oxidative stress induced by 400 mu M H2O2 can up-regulate the expression of BMP2, GATA4, MEF2C and Tbx5 in H9c2 cardiomyocytes and induce the hyperacetylation of histone H3.
(3) BMPs signaling pathway inhibitor DM has a certain antagonistic effect on the high acetylation of the histone H3 and the up regulation of the expression of GATA4 and Tbx5 induced by oxidative stress caused by 400 M H2O2, indicating that BMPs (BMP2 and other BMPs subtypes) involved in the high acetylation of histones in cardiac myocytes and the high expression of GATA4 and proteins induced by oxidative stress. In the pathological state of oxidative stress, BMPs may also be part of the upstream signaling pathway in the acetylation of cardiac myocyte histone, suggesting that oxidative stress may activate the BMPs subtype of up regulation of Tbx5 expression.
(4) BMPs signaling pathway inhibitor DM can induce the increase of MEF2C expression in H9c2 cardiomyocytes, suggesting that the comprehensive effects of BMPs subtypes in H9c2 cardiomyocytes may inhibit the expression of MEF2C, suggesting that oxidative stress may regulate the expression of MEF2C through other signaling pathways.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R725.4
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