晚期糖基化终产物对心肌细胞线粒体自噬和老化的影响及其机制研究

发布时间：2018-01-19 08:20

本文关键词： 晚期糖基化终产物心肌老化线粒体自噬 RAGE PINK1　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:衰老的过程中,损伤随机发生并逐渐积累,是一个无法避免的生理现象。当今社会老龄人口不断增加,年龄相关的退行性疾病如心血管疾病已然成为导致老年人群死亡的主要原因之一。晚期糖基化终产物(advanced glycation end products,AGEs),是指蛋白质、脂质、核酸等大分子与还原糖类如葡萄糖经过非酶糖基化反应生成的一组异质性大分子。机体内AGEs的缓慢蓄积是引起衰老的主要机制之一,在多种衰老相关性疾病的发生发展中发挥关键性作用。在心血管疾病的众多危险因素中,心肌老化是独立效应因子。在心肌组织中,AGEs 一方面可以修饰大分子和核酸,使细胞外基质蛋白发生不可逆交联,在心肌间质沉积发生纤维化从而导致心肌僵硬度增加;另一方面,它还可以通过与其特异性细胞表面受体(receptor for AGEs,RAGE),或通过直接作用等方式激活细胞内部多条信号通路,促发炎症反应、生成活性氧类物质、细胞自噬或凋亡等病理变化,导致心脏结构和功能改变,出现心肌舒张功能减退。在衰老的过程中伴随另外一个现象的发生:线粒体自噬,即机体靶向降解受损的或衰老的线粒体的过程。线粒体自噬系统的异常参与多种老化相关性疾病,诸如心血管疾病、糖尿病、阿尔兹海默症、帕金森综合征、亨廷顿氏症等。有研究表明,PINK1/Parkin信号通路是哺乳类动物细胞中调节线粒体自噬的经典途径,参与神经退行性疾病的发病过程。我们阅读文献发现衰老和线粒体自噬存在重叠的信号通路及调节分子,但是线粒体自噬在老化过程中扮演的具体角色尚不明确。诸多文献表明在多种细胞中,AGEs可以激活线粒体自噬,并且抑制其特异性受体RAGE后能显著减少自噬溶酶体的形成。但是,在AGEs诱导的心肌细胞老化的过程中,AGEs激活的线粒体自噬发挥怎样的作用以及其具体分子机制如何鲜有报道。综上所述,深入研究PINK1/Parkin介导的线粒体自噬对AGEs诱导的心肌细胞老化的影响及可能机制有重大临床意义。目的:探讨晚期糖基化终产物诱导心肌细胞线粒体自噬的机制及其在心肌细胞老化过程中的作用。方法:用AGEs干预新生乳大鼠心肌细胞后,通过β-半乳糖苷酶染色和Western blotting分别检测β-半乳糖苷酶活性和老化相关蛋白p16的表达,通过Western blotting检测线粒体自噬相关蛋白PINK1、Parkin、LC3和p62的表达水平。使用线粒体自噬抑制剂环孢霉素A(CsA)及PINK1小干扰RNA(siRNA)干预心肌细胞,从而研究线粒体自噬在AGEs诱导的心肌细胞老化中的作用。结果:与正常对照组相比,AGEs干预的心肌细胞表现出β-半乳糖苷酶染色阳性率明显增高,p16蛋白表达量显著增加。同时AGEs以剂量依赖的方式,增加了心肌细胞内PINK1和Parkin的蛋白表达水平以及LC3-Ⅱ/LC3-Ⅰ的比值。而与此伴随的是p62表达量的显著下降。给予CsA干预以及siRNA敲降PINK1后,与AGEs干预组相比,PINK1、Parkin的表达量以及LC3-Ⅱ/LC3-Ⅰ的比值明显降低,并且显著减轻由AGEs引起的SA-β-Gal活性增强和p16水平的上调。结论:PINK1/Parkin介导的线粒体自噬参与了 AGEs诱导的心肌老化过程,减轻线粒体自噬的活性可能阻止心肌细胞的老化。
[Abstract]:Background: the process of aging, damage and random accumulation, is an inevitable physiological phenomenon in today's society. The aging population growing, age-related degenerative diseases such as cardiovascular disease has become one of the major causes of death in elderly people. Advanced glycation end products (advanced glycation end products, AGEs) that refers to the protein, lipid, nucleic acid and reducing sugars such as glucose after a group of heterogeneous formation of non enzymatic glycation reaction of large molecules in the body. The slow accumulation of AGEs is one of the main mechanisms of aging, play a key role in the development and progression of many age-related diseases. In many dangerous factor of cardiovascular disease, myocardial aging is the independent effect factor. In myocardial tissue, AGEs can modify large molecules and nucleic acid, the extracellular matrix protein occurs irreversible In the cross-linking, myocardial interstitial fibrosis deposition occurred resulting in increased myocardial stiffness; on the other hand, it can also through its specific cell surface receptors (receptor, for, AGEs, RAGE) activation of multiple signaling pathways inside the cell or by direct effect, promote the inflammatory reaction, generation of reactive oxygen species, autophagy apoptosis and pathological changes, leading to the changes of cardiac structure and function, myocardial diastolic dysfunction. Another phenomenon occurs in the process of aging: mitochondrial autophagy, the process in which the body is targeted degradation of damaged or aging mitochondria. The abnormal mitochondrial autophagy system is involved in many aging related diseases such as cardiovascular disease, diabetes,, Alzheimer's disease, Parkinson syndrome, Huntington's disease. Studies have shown that PINK1/Parkin signaling pathway is self regulating mitochondrial mammalian animal cells The classical pathway of autophagy, is involved in the pathogenesis of neurodegenerative diseases. We read the literature found that aging and mitochondrial autophagy pathway are overlapping and regulating molecules, but the specific role of mitochondrial autophagy plays in the aging process is not clear. Many literatures show that in many kinds of cells, AGEs can inhibit the activation of mitochondrial autophagy, and specific receptor RAGE can significantly reduce the formation of autophagolysosomes. However, in the process of AGEs induced myocardial cell aging, mitochondrial autophagy activation of AGEs role and the molecular mechanism of how rarely reported. In summary, a clinically significant effect of mitochondrial PINK1/Parkin mediated autophagy research on aging induced by AGEs the myocardial cells and the possible mechanism. Objective: To explore the mechanism of advanced glycation end products induce mitochondrial autophagy and its In the role of myocardial cell aging. Methods: the myocardial cells of newborn rats with AGEs after intervention by beta galactosidase staining and Western blotting were used to detect the beta galactosidase activity and aging related protein p16 expression by Western blotting detection of mitochondrial autophagy related protein PINK1, Parkin, and LC3 levels p62. The use of mitochondrial autophagy inhibitor cyclosporin A (CsA) and PINK1 small interfering RNA (siRNA) to study the intervention of myocardial cells, mitochondrial autophagy in AGEs induced myocardial cell aging in rats. Results: compared with normal control group, AGEs intervention of myocardial cells showed beta galactosidase staining the positive rate increased significantly, the expression of p16 protein increased significantly. At the same time AGEs in a dose dependent manner, increasing the intracellular PINK1 and Parkin protein expression level and ratio of LC3- II /LC3- II. With this The expression of p62 was significantly decreased the amount of intervention with CsA and siRNA. After knocking down PINK1, compared with AGEs group PINK1, the expression of Parkin and LC3- II /LC3- I ratio is significantly reduced, and significantly reduce the enhancement caused by the AGEs SA- beta -Gal activity and p16 level increased. Conclusion: mitochondrial autophagy involved in the PINK1/Parkin mediated AGEs induced myocardial aging, aging reduced mitochondrial autophagy activity may prevent myocardial cells.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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