BMP2對(duì)H9c2心肌細(xì)胞組蛋白乙�；揎椀恼{(diào)控作用及其機(jī)制研究
發(fā)布時(shí)間：2018-07-31 07:07
【摘要】：目的 構(gòu)建BMP2過(guò)表達(dá)的心肌細(xì)胞模型，檢測(cè)BMP2對(duì)心臟核心轉(zhuǎn)錄因子GATA4、MEF2C和Tbx5表達(dá)的影響，并研究BMP2對(duì)H9c2心肌細(xì)胞總組蛋白H3、基因啟動(dòng)子區(qū)組蛋白H3乙�；揎椉敖M蛋白乙�；福℉ATs）亞型p300和GCN5的調(diào)控作用，以證實(shí)我們的科學(xué)假設(shè)：BMP2是心肌細(xì)胞組蛋白乙�；揎椀纳嫌涡盘�(hào)通路之一。 方法 （1）過(guò)表達(dá)BMP2的腺病毒（AdBMP2）及對(duì)照空腺病毒（AdGFP）在HEK293細(xì)胞中擴(kuò)增后，分別以不同滴度轉(zhuǎn)染大鼠H9c2心肌細(xì)胞，24h后熒光倒置顯微鏡下觀察AdBMP2/AdGFP腺病毒轉(zhuǎn)染細(xì)胞綠色熒光蛋白的表達(dá)情況，流式細(xì)胞術(shù)檢測(cè)AdBMP2/AdGFP腺病毒轉(zhuǎn)染效率。 （2）AdBMP2/AdGFP腺病毒轉(zhuǎn)染H9c2心肌細(xì)胞24h、48h和72h后收集細(xì)胞，提取mRNA，Real-Time qRT-PCR檢測(cè)各處理組細(xì)胞BMP2、MEF2C、GATA4和Tbx5及HATs亞型p300和GCN5的mRNA表達(dá)水平，篩選最佳干預(yù)時(shí)間。 （3）AdBMP2/AdGFP腺病毒轉(zhuǎn)染H9c2心肌細(xì)胞48h后收集細(xì)胞，比色法檢測(cè)各處理組H9c2心肌細(xì)胞核蛋白HATs活性，Western-blotting檢測(cè)各處理組細(xì)胞總組蛋白H3的乙�；�，ChIP-Real-Time qPCR檢測(cè)各處理組細(xì)胞MEF2C、GATA4和Tbx5啟動(dòng)子區(qū)組蛋白H3乙�；�。 結(jié)果 （1）AdBMP2轉(zhuǎn)染H9c2心肌細(xì)胞24h后，熒光倒置顯微鏡下可見細(xì)胞中出現(xiàn)大量綠色熒光，流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示AdBMP2轉(zhuǎn)染效率可達(dá)90%以上。 （2）AdBMP2轉(zhuǎn)染H9c2心肌細(xì)胞24h、48h、72h后，BMP2及心臟核心轉(zhuǎn)錄因子MEF2C和GATA4的mRNA表達(dá)水平較對(duì)照組明顯升高（P0.05），并于轉(zhuǎn)染后48h達(dá)到高峰，而Tbx5的表達(dá)沒(méi)有明顯變化。 （3）AdBMP2轉(zhuǎn)染H9c2心肌細(xì)胞48h后，HATs亞型p300的mRNA表達(dá)水平較對(duì)照組升高（P0.05），而GCN5的表達(dá)水平與對(duì)照組相比沒(méi)有明顯的變化。 （4）AdBMP2轉(zhuǎn)染H9c2心肌細(xì)胞48h后，細(xì)胞核蛋白HATs活性較對(duì)照組明顯升高（P0.05），總組蛋白H3乙�；揭草^對(duì)照組明顯上調(diào)（P0.05），MEF2C、GATA4啟動(dòng)子區(qū)組蛋白H3乙�；揭嘞鄳�(yīng)升高（P0.05），但Tbx5啟動(dòng)子區(qū)組蛋白H3乙�；脚c對(duì)照組相比未見明顯變化。 結(jié)論 （1）BMP2可上調(diào)H9c2心肌細(xì)胞中心臟核心轉(zhuǎn)錄因子MEF2C和GATA4的表達(dá)，，而對(duì)Tbx5的表達(dá)沒(méi)有影響。 （2）BMP2可引起H9c2心肌細(xì)胞組蛋白H3高乙�；�，可能是H9c2心肌細(xì)胞組蛋白乙�；揎椀纳嫌涡盘�(hào)通路之一。 （3）BMP2引起的H9c2心肌細(xì)胞組蛋白H3高乙�；赡芘cHATs亞型p300有關(guān)。 （4）BMP2對(duì)MEF2C和GATA4啟動(dòng)子區(qū)組蛋白H3乙�；拇龠M(jìn)作用可能是BMP2引起MEF2C和GATA4表達(dá)上調(diào)的機(jī)制之一，而Tbx5啟動(dòng)子區(qū)組蛋白H3乙�；词蹷MP2的影響可能是Tbx5的表達(dá)不受BMP2調(diào)控的原因之一。 目的 使用HATs亞型p300的抑制劑姜黃素（Curcumin）來(lái)研究p300在BMP2致H9c2心肌細(xì)胞心臟核心轉(zhuǎn)錄因子GATA4和MEF2C高表達(dá)及組蛋白高乙�；械淖饔�，以證實(shí)我們的科學(xué)假設(shè)：p300參與BMP2對(duì)H9c2心肌細(xì)胞心臟核心轉(zhuǎn)錄因子表達(dá)及組蛋白乙�；揎椀恼{(diào)控作用。 方法 （1）AdBMP2轉(zhuǎn)染H9c2心肌細(xì)胞后，用不同濃度的p300HAT活性抑制劑姜黃素(10μM,20μM,30μM,40μM)處理細(xì)胞(6h,12h,24h,48h)，比色法檢測(cè)各處理組細(xì)胞HATs活性，篩選姜黃素最佳處理濃度及時(shí)間。 （2）AdBMP2和/或姜黃素處理H9c2心肌細(xì)胞后，Real-Time qRT-PCR檢測(cè)各處理組細(xì)胞心臟核心轉(zhuǎn)錄因子GATA4、MEF2C和Tbx5及HATs亞型p300和GCN5的表達(dá)水平，Western-blotting檢測(cè)各處理組細(xì)胞總組蛋白H3的乙�；�，ChIP-Real-Time qPCR檢測(cè)各處理組細(xì)胞GATA4、MEF2C和Tbx5啟動(dòng)子區(qū)組蛋白H3乙�；�。 結(jié)果 （1）不同濃度（10μM,20μM,30μM,40μM)的姜黃素處理細(xì)胞24h后，H9c2心肌細(xì)胞的HATs活性明顯下降（P0.05），并于40μM達(dá)到最低點(diǎn)。40μM姜黃素分別處理細(xì)胞6h,12h,24h,48h后，HATs活性于處理后12h達(dá)到最低點(diǎn)。 （2）AdBMP2和姜黃素共同處理H9c2心肌細(xì)胞后，心臟核心轉(zhuǎn)錄因子GATA4和MEF2C及HATs亞型p300的mRNA表達(dá)水平較單獨(dú)AdBMP2處理組明顯下降（P0.05），而Tbx5及GCN5的mRNA表達(dá)水平在各處理組之間沒(méi)有明顯的變化。 （3）AdBMP2與姜黃素共同處理H9c2心肌細(xì)胞后，細(xì)胞中總組蛋白H3乙�；郊癎ATA4和MEF2C啟動(dòng)子區(qū)組蛋白H3乙�；捷^單獨(dú)AdBMP2處理組明顯下降（P0.05），而Tbx5啟動(dòng)子區(qū)組蛋白H3乙�；诟魈幚斫M之間沒(méi)有明顯的變化。 結(jié)論 （1）姜黃素可抑制HATs亞型p300的表達(dá)，而對(duì)GCN5的表達(dá)沒(méi)有影響。 （2）在H9c2心肌細(xì)胞中，p300HAT活性抑制劑姜黃素可拮抗BMP2引起的組蛋白H3高乙�；癎ATA4和MEF2C的高表達(dá)，說(shuō)明p300參與BMP2對(duì)H9c2心肌細(xì)胞GATA4和MEF2C表達(dá)及組蛋白乙�；恼{(diào)控。 （3）在H9c2心肌細(xì)胞中，Tbx5啟動(dòng)子區(qū)組蛋白H3乙�；捌浔磉_(dá)可能不受p300的調(diào)控。 目的 使用BMPs信號(hào)通路抑制劑dorsomorphin（DM）來(lái)研究BMPs在氧化應(yīng)激反應(yīng)所致H9c2心肌細(xì)胞組蛋白高乙�；械淖饔�，以證實(shí)我們的科學(xué)假設(shè)：BMPs參與介導(dǎo)氧化應(yīng)激反應(yīng)所致H9c2心肌細(xì)胞組蛋白高乙�；�。 方法 （1）不同濃度H2O2(50μM、100μM、150μM、200μM、250μM、300μM、350μM、400μM)處理H9c2心肌細(xì)胞，24h后采用MTT法檢測(cè)各處理組細(xì)胞的存活率。 （2）5μM的BMPs信號(hào)通路抑制劑DM和/或適宜濃度的H2O2處理細(xì)胞，Real-Time qRT-PCR檢測(cè)各處理組細(xì)胞BMP2及心臟核心轉(zhuǎn)錄因子GATA4，MEF2C和Tbx5的表達(dá)水平，Western-blotting檢測(cè)各處理組細(xì)胞總組蛋白H3的乙�；�。 結(jié)果 （1）50、100和150μM濃度的H2O2對(duì)H9c2心肌細(xì)胞的存活率沒(méi)有明顯的影響，200、250、300、350、400和450μM濃度的H2O2處理H9c2心肌細(xì)胞后，細(xì)胞的存活率分別降低10.5％、16.9％、21.9％、32.4％、47.0％和58.6％。 （2）400μM H2O2處理H9c2心肌細(xì)胞后，BMP2、GATA4、MEF2C和Tbx5的mRNA表達(dá)水平較空白對(duì)照組明顯升高（P0.05），細(xì)胞組蛋白H3的乙�；揭嘞鄳�(yīng)升高（P0.05）。 （3）400μM H2O2和DM共同處理H9c2心肌細(xì)胞后，H9c2心肌細(xì)胞中總組蛋白H3乙�；捷^單獨(dú)400μM H2O2處理組有所下降（P0.05），GATA4和Tbx5的mRNA表達(dá)水平也較單獨(dú)400μMH2O2處理組有所降低（P0.05），而MEF2C的mRNA表達(dá)水平較單獨(dú)400μM H2O2處理組有所升高（P0.05）。 結(jié)論 （1）利用H2O2成功構(gòu)建H9c2心肌細(xì)胞氧化損傷模型。 （2）400μM H2O2引起的氧化應(yīng)激可上調(diào)H9c2心肌細(xì)胞BMP2、GATA4、MEF2C和Tbx5的表達(dá)并引起細(xì)胞組蛋白H3高乙�；�。 （3）BMPs信號(hào)通路抑制劑DM對(duì)400μM H2O2引起的氧化應(yīng)激所致H9c2心肌細(xì)胞組蛋白H3高乙�；癎ATA4和Tbx5表達(dá)上調(diào)具有一定的拮抗作用，說(shuō)明BMPs（BMP2及其它BMPs亞型）參與介導(dǎo)氧化應(yīng)激引起的心肌細(xì)胞組蛋白高乙�；癎ATA4和Tbx5的高表達(dá)，提示在氧化應(yīng)激的病理狀態(tài)下，BMPs可能也是心肌細(xì)胞組蛋白乙�；揎椛嫌涡盘�(hào)通路的組成部分，也提示氧化應(yīng)激可能激活了上調(diào)Tbx5表達(dá)的BMPs亞型。 （4）BMPs信號(hào)通路抑制劑DM可引起H9c2心肌細(xì)胞MEF2C的表達(dá)升高，提示在H9c2心肌細(xì)胞中，BMPs各亞型的綜合效應(yīng)可能是對(duì)MEF2C的表達(dá)起抑制作用，也提示氧化應(yīng)激可能通過(guò)其它信號(hào)通路調(diào)控MEF2C的表達(dá)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R725.4
本文編號(hào)：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R725.4
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