發(fā)布時間：2018-08-30 19:30

【摘要】：第1章引言急性心肌梗塞在心血管疾病中發(fā)病率和死亡率極高,缺血/再灌注損傷仍是急性心肌梗塞臨床治療中面臨的難題。如何減輕缺血/再灌注損傷是心肌損傷與保護的研究熱點。研究心肌損傷發(fā)生機制,將有助于我們?yōu)榕R床防治心肌缺血損傷提供理論和實踐依據(jù)。因此,我們基于lncRNA-miRNA-Notch1調控假說,建立心肌細胞缺氧/復氧損傷及自噬保護模型,通過高通量測序技術及生物信息學比較分析,篩選出在自噬保護中具有調控Notch1蛋白作用的miRNA,以及具有調控該miRNA的lncRNA,為研究Notch1信號通路在自噬性心肌保護的分子機制提供科學依據(jù)。第2章Notch1信號通路增強自噬對抗心肌缺氧復氧損傷的保護作用目的:篩選有效調控自噬的3-MA及RAPA試劑濃度,以及探討Notch1信號通路在自噬對抗心肌缺氧復氧損傷中的作用。方法:建立心肌細胞損傷模型,給予3-MA、RAPA干預,檢測細胞活力,篩選3-MA及RAPA有效試劑濃度;利用Ad-N1ICD及Ad-N1ICD-shRNA干預Notch1信號通路,檢測H/R損傷中心肌細胞活力情況。結果:本研究中3-MA和RAPA最佳試劑濃度分別為1μM和50nM;Notch1過表達可提高H/R的心肌細胞活力(p0.01),干擾Notch1可明顯降低H/R心肌細胞活力(p0.05);3-MA加入Notch1過表達組中,H/R的心肌細胞活力再次降低(p0.01),RAPA加入Notch1干擾組中,H/R的心肌細胞活力恢復(p0.01)。結論:Notch1信號通路可增強自噬對抗心肌缺氧復氧損傷發(fā)揮保護作用。第3章高通量測序及生物信息學篩選自噬性心肌保護中Notch1信號通路相關miRNA及l(fā)ncRNA目的:借助高通量測序及生物信息學比較分析篩選在自噬性心肌保護中具有調控Notch1信號通路的關鍵miRNA及l(fā)ncRNA。方法:提取心肌缺氧/復氧損傷中的總RNA,借助高通量測序及生物信息學分析系統(tǒng),篩選具有差異表達的miRNA及l(fā)ncRNA。結果:篩選出了可能調控Notch1基因表達的miR-702-5p、miR-344g、miR-323-5p、miR-6334、miR-5132-5p、miR-15b-5p、miR-3562;生物信息學分析發(fā)現(xiàn)lncRNA-4321可與miR-702-5p直接結合并影響其活性;在RAPA和3-MA處理心肌細胞中,Realtime-PCR檢測顯示lncRNA-4321表達量與心肌自噬成正比,miR-702-5p表達量與心肌自噬成反比。結論:lncRNA-4321/miR-702-5p/Notch1可能在自噬性心肌保護中發(fā)揮重要作用。
[Abstract]:Chapter 1 introduces that the incidence and mortality of acute myocardial infarction in cardiovascular disease is very high. Ischemia / reperfusion injury is still a difficult problem in the clinical treatment of acute myocardial infarction. How to reduce ischemia / reperfusion injury is the focus of myocardial injury and protection. Studying the mechanism of myocardial injury will help us to provide theoretical and practical basis for clinical prevention and treatment of myocardial ischemia injury. Therefore, based on the lncRNA-miRNA-Notch1 regulation hypothesis, we established a cardiomyocyte model of hypoxia / reoxygenation injury and autophagy protection, and analyzed it by high-throughput sequencing and bioinformatics. The screening of miRNA, which has the function of regulating Notch1 protein in autophagy protection and lncRNA, which has the function of regulating miRNA provide scientific basis for studying the molecular mechanism of Notch1 signaling pathway in autophagy myocardial protection. Chapter 2 Notch1 signaling pathway enhances the protective effect of autophagy on myocardial hypoxic-reoxygenation injury objective: to screen the concentration of 3-MA and RAPA reagents that can effectively regulate autophagy and to explore the role of Notch1 signaling pathway in anti-hypoxia and reoxygenation injury of myocardium. Methods: myocardial injury model was established, 3-MARAPA was given to detect cell viability, 3-MA and RAPA effective reagent concentrations were screened, and Notch1 signaling pathway was interfered by Ad-N1ICD and Ad-N1ICD-shRNA to detect myocardial cell viability in H / R injury. Results: the best concentration of 3-MA and RAPA in this study was 1 渭 M and 50nM respectively, which increased the viability of H / R cardiomyocytes (p0.01). Interfering with Notch1 could significantly reduce the activity of H / R cardiomyocytes (p0.05) 3-MA added to Notch1 overexpression group. The secondary decrease (p0.01) and the activity recovery of H / R in the Notch1 interference group (p0.01). Conclusion the fraction Notch1 signaling pathway can enhance the protective effect of autophagy on myocardial hypoxia and reoxygenation injury. Chapter 3 High-throughput sequencing and Bioinformatics screening Notch1 signaling Path-Related miRNA and lncRNA in autophagy Myocardial Protection objective: to regulate Notch1 in autophagic myocardial protection by high-throughput sequencing and bioinformatics comparative analysis Key miRNA and lncRNA. of signal Pathway Methods: total RNA, from myocardial hypoxia / reoxygenation injury was extracted by high throughput sequencing and bioinformatics analysis system to screen miRNA and lncRNA. with differential expression. Results: the bioinformatics analysis of miR-702-5p,miR-344g,miR-323-5p,miR-6334,miR-5132-5p,miR-15b-5p,miR-3562;, which could regulate the expression of Notch1 gene, found that lncRNA-4321 could directly bind to miR-702-5p and affect its activity. In RAPA and 3-MA treated cardiomyocytes, the expression of lncRNA-4321 was directly proportional to myocardial autophagy and the expression of miR-702-5p was inversely proportional to myocardial autophagy. Conclusion: 1: lncRNA-4321% miR-702-5p / Notch1 may play an important role in the protection of autophagic myocardium.
【學位授予單位】：南昌大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R54

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