本文選題：心肌重塑 + 競爭性內源RNA��； 參考：《哈爾濱工業(yè)大學》2016年博士論文

【摘要】：心血管疾病是危害人類健康的主要疾病之一,給社會造成巨大的經(jīng)濟負擔。深入研究心血管疾病發(fā)病的分子機制并在此基礎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,是生命科學需要解決的重大基礎科學問題。壓力過負荷以及高糖等因素引起心肌重塑導致心肌結構和功能發(fā)生變化,促進心血管疾病的發(fā)生。要找到應對不同環(huán)境下心臟重塑和功能失衡的有效方法,當前最重要的是要研究了解心肌重塑變化發(fā)生的分子機制。觸發(fā)心肌重塑的反應與多種信號的激活有關,其中包括轉錄后調控和翻譯后調控兩個方面,PTEN和CKIP-1是信號轉導途徑中重要的節(jié)點分子,本文以PTEN和CK IP-1為目標分子,分別從信號分子轉錄后調控和翻譯后調控兩個層面,研究不同狀態(tài)下心肌重塑的分子機制。在心肌重塑的轉錄后調控機制研究方面,本文通過在糖尿病小鼠心肌中對預測的PTEN的競爭性內源RNA(competing endogenous RNAs,ce RNAs)進行了小規(guī)模的篩選,并在兩種不同類型的糖尿病小鼠心肌中進行表達驗證,獲得在糖尿病小鼠心肌中表達最顯著的PTEN ce RNA-DKK1,同時利用siRNA敲低和過表達的實驗驗證DKK1對PTEN 3′非翻譯區(qū)結合miRNA能力具有調控作用,通過PTEN 3′非翻譯區(qū)的熒光素酶報告實驗研究證實PTEN ce RNA-DKK1對PTEN的調控依賴于miRNA的作用;發(fā)現(xiàn)PTEN和DKK1的mRNA受共有miRN A的調控,證實DKK 1對PTEN具有競爭性內源RN A的調控作用,并且該調控機制對高糖誘導的心肌重塑具有影響,DKK1的敲低可以抑制高糖導致的心肌細胞凋亡增加和葡萄糖攝取能力的減弱,并且DKK1對心肌重塑的調控作用是依賴于PI3K/Akt信號通路的。從而證實DKK1對PTEN的競爭性內源RN A調控機制對高糖誘導的心肌重塑發(fā)揮了重要的調控作用,從轉錄后調控研究方面闡釋了高糖誘導心肌重塑的分子機制。通過構建主動脈縮窄所致壓力過負荷引起的小鼠心肌重塑模型,免疫組織化學染色和Western blotting以及實時定量PCR方法檢測心肌重塑發(fā)生過程中CKIP-1 mRN A和蛋白的表達情況,并利用心肌肥大病人的心肌組織樣本,發(fā)現(xiàn)CKIP-1在心肌重塑發(fā)生早期出現(xiàn)代償性升高,而在后期發(fā)生表達降低的情況,證實CKIP-1和心肌重塑密切相關;并利用CKIP-1敲除的模式小鼠,通過心臟組織形態(tài)學結果,心肌胚胎期基因表達情況和小動物超聲心動技術分析其不同年齡階段心臟的表型,發(fā)現(xiàn)CKIP-1敲除可促進心肌重塑,引起心肌肥大和心臟功能下降;利用CKIP-1敲除的模式小鼠,構建主動脈縮窄所致壓力過負荷引起的小鼠心肌重塑模型,發(fā)現(xiàn)CKIP-1敲除可顯著促進主動脈縮窄所致壓力過負荷引起的心肌重塑和心臟功能下降,證實CKIP-1敲除促進壓力過負荷誘發(fā)的心肌重塑;通過構建CKIP-1心肌特異轉基因小鼠模型,發(fā)現(xiàn)CKIP-1轉基因可顯著抑制壓力過負荷引起的心肌重塑和和心臟功能下降,確認CKIP-1對心肌重塑的重要調控作用。在心肌重塑的翻譯后調控機制研究方面,通過蛋白質譜分析,GST-pull down實驗和不同條件下的蛋白免疫共沉淀實驗,首次證實CK IP-1與HDAC4等IIa型HDACs成員之間的相互作用;通過熒光素酶報告實驗,證實CKIP-1對HDAC4下游關鍵轉錄因子MEF2轉錄活性的調控作用,并通過siRNA敲低的實驗,證實CKIP-1通過HDAC4發(fā)揮了對MEF2轉錄活性的調控作用;通過細胞定位的實驗,以及CKIP-1小鼠和心肌特異轉基因小鼠的心肌組織切片,證實CKIP-1對HDAC4的細胞定位具有重要調控作用;通過對HDAC4磷酸化水平的分析,證實CKIP-1通過對HDAC4磷酸化水平的影響而調控HDAC4的細胞定位;并且發(fā)現(xiàn)CKIP-1通過與HDAC4和PP2AC之間的相互作用,促進了PP2AC與HDAC 4之間的相互作用,對HDAC4的去磷酸化具有了重要的調節(jié)作用,從而調控了HDAC4的細胞定位和MEF2轉錄活性,最終對心肌重塑發(fā)揮重要的調控作用。從翻譯后調控-蛋白去磷酸化的角度,證實CKIP-1對心肌重塑的重要調控作用。綜上所述,在轉錄后調控研究方面,本文發(fā)現(xiàn)競爭性內源RNA作用機制對高糖誘導的心肌重塑發(fā)揮了重要調控作用,在翻譯后調控研究方面,發(fā)現(xiàn)CKIP-1通過影響PP2A對HDAC4的去磷酸化作用對壓力過負荷引起的心肌重塑發(fā)揮了調控作用。進一步闡明了不同因素誘導心肌重塑的分子機制。
[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.
【學位授予單位】：哈爾濱工業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R54

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