本文選題：H9c2心肌細胞 + BMP信號通路�。� 參考：《重慶醫(yī)科大學》2014年碩士論文

【摘要】：目的： 采用Bone morphogenetic protein（骨形態(tài)形成蛋白，BMP）信號通路的特異性抑制劑dorsomorphin（DM）阻斷其傳導，以證實BMP信號通路介導酒精致組蛋白高乙�；险{心臟核心轉錄因子表達的科學假說。 方法： （1）不同濃度酒精濃度（0mM、10mM、50mM、100mM、200mM、500mM和1000mM）處理H9c2細胞，36h后采用MTT檢測各干預組H9c2細胞存活率，篩選最適酒精干預濃度。 （2）酒精干預細胞36h后采用Real-Time qRT-PCR方法檢測BMPs各亞型mRNA的表達水平；加入不同DM濃度（0μM、1μM、2.5μM、5μM、10μM和20μM）阻斷酒精作用，36h后采用Real-TimeqRT-PCR方法檢測BMPs通路下游靶基因GATA4mRNA表達水平，，篩查出最適DM濃度。 （3）酒精和/或DM處理細胞36h后，應用Real-Time qRT-PCR技術檢測心臟核心轉錄因子MEF2C、GATA4、Nkx2.5和Tbx5的表達，及Smad4mRNA的表達水平；采用Western-blotting技術檢測H9c2細胞組蛋白H3總乙�；胶托呐K發(fā)育相關基因Cx43表達水平；ChIP-Real-Time qPCR技術檢測核心轉錄因子啟動子區(qū)域組蛋白H3乙�；�。 結果： （1）10mM、50mM、100mM酒精干預36h后H9c2細胞生存率與對照組比較沒有變化（P＞0.05），200mM、500mM和1000mM酒精使細胞生存率降低25.4%、37.0%和75.0%（P＜0.05）。 （2）100mM酒精干預36h后，H9c2心肌細胞BMP2（1.60±0.14）、BMP4（1.27±0.08）、BMP6（1.44±0.25）、BMP7（1.59±0.06）mRNA相對表達量均較對照組升高（P＜0.05）；BMP5（1.14±0.08）、BMP10（1.05±0.09）mRNA相對表達量較對照組有升高趨勢，但差異無統(tǒng)計學意義（P＞0.05）。 （3）100mM酒精和不同濃度DM干預后，GATA4mRNA的表達呈現(xiàn)出先降低后上升的趨勢，且在DM濃度為5μM時達到最低。 （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表達水平升高（P0.05）；加入5μM DM后能降至正常水平（P0.05）；5μMDM可使MEF2C mRNA表達水平上升1.2倍（P0.05）；100mM酒精使Cx43蛋白表達水平升高2.7倍（P0.05）；加入5μM DM后能降至正常水平（P0.05）；100mM酒精、5μM DM對TBX5mRNA表達（0.98±0.08）、（0.95±0.23）無明顯影響（P0.05）。 （5）100mM酒精可使組蛋白H3總乙�；缴仙�2.4倍（P0.05），加入5μM DM后降至對照組水平（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）啟動子區(qū)域的組蛋白H3乙�；缴撸≒0.05），加入5μM DM后MEF2C和GATA4啟動子區(qū)域乙�；较陆抵琳＃≒0.05），Nkx2.5啟動子區(qū)域乙酰化水平下調（1.25±0.16），但未降至正常（P0.05）。100mM酒精、5μMDM未對TBX5啟動子區(qū)域組蛋白H3乙�；�0.81±0.35）（、1.09±0.36）產生明顯影響（P0.05）。 結論 （1）酒精可以引起與心臟相關的BMP亞型mRNA表達上升。 （2）BMP信號通路介導酒精對MEF2C、GATA4和Nkx2.5啟動子區(qū)組蛋白H3乙�；拇龠M作用可能是酒精引起MEF2C、 GATA4和Nkx2.5表達上調的機制之一。
[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 鹵0.06)mRNA in H9c2 cardiomyocytes was significantly higher than that in the control group (1.44 鹵0.25mM, 1.44 鹵0.25mm). The relative expression of BMP51.14 鹵0.08BMP100.05 鹵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 鹵0. 15 mRNA and 1. 41 鹵0. 18) and Smad4(1.78 鹵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 鹵0.08 (0.95 鹵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 鹵0.24 Gata 42.04 鹵0.69) and Nkx2.5(1.50 鹵0.17) promoter region was increased by 100mm alcohol. The acetylation level of MEF2C and GATA4 promoter region decreased to 1.25 鹵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 鹵0.35) (1.09 鹵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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R725.4

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