高糖和压力过负荷诱导心肌重塑的调控机制研究

发布时间：2018-06-25 04:26

本文选题：心肌重塑 + 竞争性内源RNA　；参考：《哈尔滨工业大学》2016年博士论文

【摘要】：心血管疾病是危害人类健康的主要疾病之一,给社会造成巨大的经济负担。深入研究心血管疾病发病的分子机制并在此基础上建立新的防治策略和防治措施,降低心血管疾病的死亡率,是生命科学需要解决的重大基础科学问题。压力过负荷以及高糖等因素引起心肌重塑导致心肌结构和功能发生变化,促进心血管疾病的发生。要找到应对不同环境下心脏重塑和功能失衡的有效方法,当前最重要的是要研究了解心肌重塑变化发生的分子机制。触发心肌重塑的反应与多种信号的激活有关,其中包括转录后调控和翻译后调控两个方面,PTEN和CKIP-1是信号转导途径中重要的节点分子,本文以PTEN和CK IP-1为目标分子,分别从信号分子转录后调控和翻译后调控两个层面,研究不同状态下心肌重塑的分子机制。在心肌重塑的转录后调控机制研究方面,本文通过在糖尿病小鼠心肌中对预测的PTEN的竞争性内源RNA(competing endogenous RNAs,ce RNAs)进行了小规模的筛选,并在两种不同类型的糖尿病小鼠心肌中进行表达验证,获得在糖尿病小鼠心肌中表达最显著的PTEN ce RNA-DKK1,同时利用siRNA敲低和过表达的实验验证DKK1对PTEN 3′非翻译区结合miRNA能力具有调控作用,通过PTEN 3′非翻译区的荧光素酶报告实验研究证实PTEN ce RNA-DKK1对PTEN的调控依赖于miRNA的作用;发现PTEN和DKK1的mRNA受共有miRN A的调控,证实DKK 1对PTEN具有竞争性内源RN A的调控作用,并且该调控机制对高糖诱导的心肌重塑具有影响,DKK1的敲低可以抑制高糖导致的心肌细胞凋亡增加和葡萄糖摄取能力的减弱,并且DKK1对心肌重塑的调控作用是依赖于PI3K/Akt信号通路的。从而证实DKK1对PTEN的竞争性内源RN A调控机制对高糖诱导的心肌重塑发挥了重要的调控作用,从转录后调控研究方面阐释了高糖诱导心肌重塑的分子机制。通过构建主动脉缩窄所致压力过负荷引起的小鼠心肌重塑模型,免疫组织化学染色和Western blotting以及实时定量PCR方法检测心肌重塑发生过程中CKIP-1 mRN A和蛋白的表达情况,并利用心肌肥大病人的心肌组织样本,发现CKIP-1在心肌重塑发生早期出现代偿性升高,而在后期发生表达降低的情况,证实CKIP-1和心肌重塑密切相关;并利用CKIP-1敲除的模式小鼠,通过心脏组织形态学结果,心肌胚胎期基因表达情况和小动物超声心动技术分析其不同年龄阶段心脏的表型,发现CKIP-1敲除可促进心肌重塑,引起心肌肥大和心脏功能下降;利用CKIP-1敲除的模式小鼠,构建主动脉缩窄所致压力过负荷引起的小鼠心肌重塑模型,发现CKIP-1敲除可显著促进主动脉缩窄所致压力过负荷引起的心肌重塑和心脏功能下降,证实CKIP-1敲除促进压力过负荷诱发的心肌重塑;通过构建CKIP-1心肌特异转基因小鼠模型,发现CKIP-1转基因可显著抑制压力过负荷引起的心肌重塑和和心脏功能下降,确认CKIP-1对心肌重塑的重要调控作用。在心肌重塑的翻译后调控机制研究方面,通过蛋白质谱分析,GST-pull down实验和不同条件下的蛋白免疫共沉淀实验,首次证实CK IP-1与HDAC4等IIa型HDACs成员之间的相互作用;通过荧光素酶报告实验,证实CKIP-1对HDAC4下游关键转录因子MEF2转录活性的调控作用,并通过siRNA敲低的实验,证实CKIP-1通过HDAC4发挥了对MEF2转录活性的调控作用;通过细胞定位的实验,以及CKIP-1小鼠和心肌特异转基因小鼠的心肌组织切片,证实CKIP-1对HDAC4的细胞定位具有重要调控作用;通过对HDAC4磷酸化水平的分析,证实CKIP-1通过对HDAC4磷酸化水平的影响而调控HDAC4的细胞定位;并且发现CKIP-1通过与HDAC4和PP2AC之间的相互作用,促进了PP2AC与HDAC 4之间的相互作用,对HDAC4的去磷酸化具有了重要的调节作用,从而调控了HDAC4的细胞定位和MEF2转录活性,最终对心肌重塑发挥重要的调控作用。从翻译后调控-蛋白去磷酸化的角度,证实CKIP-1对心肌重塑的重要调控作用。综上所述,在转录后调控研究方面,本文发现竞争性内源RNA作用机制对高糖诱导的心肌重塑发挥了重要调控作用,在翻译后调控研究方面,发现CKIP-1通过影响PP2A对HDAC4的去磷酸化作用对压力过负荷引起的心肌重塑发挥了调控作用。进一步阐明了不同因素诱导心肌重塑的分子机制。
[Abstract]:Cardiovascular disease is one of the major diseases that harm human health, causing great economic burden to the society. It is a major basic scientific problem to solve the molecular mechanism of cardiovascular disease and to set up new prevention and control strategies and measures to reduce the mortality of cardiovascular diseases. Cardiac remodeling leads to cardiac structure and function changes and promotes the occurrence of cardiovascular disease. It is important to study the molecular mechanism of understanding the changes of cardiac remodeling and the reaction and multiple of cardiac remodeling. The activation of the signal is related, including post transcriptional regulation and post translation regulation two aspects. PTEN and CKIP-1 are important node molecules in the signal transduction pathway. In this paper, PTEN and CK IP-1 are used as the target molecules. The molecular mechanism of myocardial remodeling in different states is studied from two levels after the regulation of signal molecules after transcriptional regulation and post translation. In the study of post transcriptional regulatory mechanism of myocardial remodeling, a small scale screening was conducted in the myocardium of diabetic mice by the competitive endogenous RNA (competing endogenous RNAs, CE RNAs) in the myocardium of the diabetic mice, and the expression was verified in the myocardium of two different types of diabetic mice to obtain the table in the myocardium of diabetic mice. The most significant PTEN CE RNA-DKK1, while using siRNA knockout and overexpressed experiments to verify that DKK1 has a regulatory effect on miRNA ability in PTEN 3 'non translation region, and through the luciferase report experimental study of the 3' non translation zone of PTEN, the regulation of PTEN CE RNA-DKK1 on PTEN is dependent on the effect. The regulation of miRN A confirms that DKK 1 has a competitive endogenous RN A regulating effect on PTEN, and that the regulatory mechanism has an effect on high glucose induced myocardial remodeling. The knock down of DKK1 can inhibit the increase of myocardial apoptosis and impaired glucose uptake by high glucose, and the regulation of DKK1 on myocardial remodeling depends on PI3K/Ak T signaling pathway. Thus it is confirmed that DKK1's competitive endogenous endogenous RN A regulatory mechanism plays an important role in high glucose induced myocardial remodeling. The molecular mechanism of high glucose induced myocardial remodeling is explained from post transcriptional regulation research. The model of myocardial remodeling in mice induced by pressure overload caused by aortic coarctation is avoided. The expression of CKIP-1 mRN A and protein in the process of myocardial remodeling was detected by immunohistochemical staining, Western blotting and real-time quantitative PCR, and the myocardial tissue samples of the patients with myocardial hypertrophy were used to detect the compensatory elevation of CKIP-1 in the early stage of myocardial remodeling, while the expression decreased in the later period and confirmed CKIP-1. It is closely related to cardiac remodeling; and using CKIP-1 knockout model mice, through cardiac histomorphological results, cardiac embryonic stage gene expression and small animal echocardiography analysis of the heart phenotype at different age stages, it is found that CKIP-1 knockout can promote myocardial remodeling, cause cardiac hypertrophy and cardiac function decline; use CKIP-1 In the knockout model mice, a model of myocardial remodeling induced by stress overload caused by coarctation of the aorta was constructed. It was found that CKIP-1 knockout could significantly promote myocardial remodeling and cardiac dysfunction caused by pressure overload caused by aortic coarctation. It was proved that CKIP-1 knockout promoted myocardial remodeling induced by stress overload; and the CKIP-1 myocardium was constructed. The model of transgenic mice found that CKIP-1 transgenic could significantly inhibit myocardial remodeling and cardiac dysfunction caused by stress overload, and confirmed the important regulatory role of CKIP-1 on myocardial remodeling. In the study of post-translational mechanism of myocardial remodeling, protein mass spectrometry analysis, GST-pull down experiment and protein immunity under different conditions The interaction between CK IP-1 and II a HDACs members, such as HDAC4, was confirmed for the first time, and the regulation of CKIP-1 on the transcriptional activity of the key transcriptional factor of the downstream HDAC4 was confirmed by the luciferase reporter experiment, and through the experiment of siRNA knock down, it was proved that CKIP-1 through HDAC4 exerts the regulation of the transcriptional activity of MEF2. Cell localization experiments, as well as the myocardial tissue sections of CKIP-1 mice and myocardium specific transgenic mice, confirm that CKIP-1 plays an important role in regulating the cell location of HDAC4. Through the analysis of the phosphorylation level of HDAC4, it is proved that CKIP-1 regulates the cell location of HDAC4 by the effect of HDAC4 phosphorylation level; and it is found that CKIP-1 passes with H. The interaction between DAC4 and PP2AC promotes the interaction between PP2AC and HDAC 4, which plays an important regulatory role in the dephosphorylation of HDAC4, thus regulates the cell location and MEF2 transcriptional activity of HDAC4, and ultimately plays an important regulatory role in the remodeling of myocardium. From the angle of post translation regulation protein dephosphorylation, CKIP-1 pairs are confirmed. The important regulatory role of myocardial remodeling. In summary, in the post transcriptional regulation study, we found that the competitive endogenous RNA mechanism plays an important role in the regulation of high glucose induced myocardial remodeling. In the post translation study, it is found that CKIP-1 affects the myocardium caused by stress overload by affecting the dephosphorylation of PP2A to HDAC4. Remodeling played a regulatory role, further elucidated the molecular mechanism of different factors inducing myocardial remodeling.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54

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