miR-98通过抑制氧化低密度脂蛋白受体-1调控动脉粥样硬化形成的机制研究

发布时间：2018-07-01 14:03

本文选题：动脉粥样硬化 + 氧化低密度脂蛋白受体1　；参考：《安徽医科大学》2017年博士论文

【摘要】：动脉粥样硬化(Atherosclerosis,AS)是心血管疾病的最主要病因,其参与多种致命性心血管疾病如:心肌缺血、心绞痛、心肌梗塞以及心力衰竭等。如何抑制延缓AS的形成,降低AS性心血管疾病的发生率和死亡率已成为该领域共同努力的目标。AS的形成机制复杂,主要包括以下因素:氧化应激,动脉内皮损伤及功能障碍,泡沫细胞形成以及后续的脂质沉积和血栓形成等。其中氧化应激、内皮功能障碍和泡沫细胞形成均为AS早期的特征性病理变化过程。微小核糖核酸(micro RNAs,mi Rs)是一种由约22个核苷酸组成的RNA小片段,通过与靶基因的信使RNA 3’端结合,有效抑制m RNA的转录与翻译,从而发挥不同的生物学效应。研究证实,mi Rs参与多种心血管疾病,如心肌缺血、心肌纤维化、高血压、糖尿病性心肌病、心力衰竭以及AS等。然而,这些研究仍处于初期阶段,mi Rs调控AS的机制如何,如何利用mi Rs治疗AS都是悬而未决的问题。前期研究结果发现,AS患者血浆中的某些mi Rs较健康志愿者表达存在明显差异。通过生物信息学预测并分析microarray数据,发现这些mi Rs具有一个共同的靶基因,氧化低密度脂蛋白受体-1(lectin-like oxidized low-density lipoprotein receptor-1,LOX-1)。LOX-1是氧化低密度脂蛋白(oxidized low density lipoprotein,Ox-LDL)在细胞膜上的受体,具有清道夫受体的特性。如前所述,AS早期主要表现为氧化应激、内皮功能障碍、泡沫细胞形成以及脂质沉积。因此,本研究针对上述特点,采用科学的筛选方法选取一个最具代表性的mi R,研究其对AS的影响,并进一步探讨其可能的机制。研究内容主要包括以下四个部分:1.AS患者与健康志愿者血浆mi RNA表达比较首先采用microarray技术检测AS患者与健康志愿者血浆mi Rs及m RNAs的表达。发现28个mi Rs以及32个m RNA表达发生明显变化。通过Real-time PCR验证结果发现mi R-98表达变化具有统计学意义。利用生物信息学软件再次验证mi R-98与LOX-1存在结合位点。LOX-1是AS形成中的重要调节因子,提示mi Rs可能通过调控LOX-1表达影响AS的形成。2.体外模拟AS环境评估mi R-98对AS形成的影响为模拟AS的体外环境,本研究使用Ox-LDL干预人主动脉内皮细胞,结果发现随着Ox-LDL浓度的逐步升高(0-10μg/m L),mi R-98的表达逐渐降低,而与此同时,LOX-1的表达逐渐增高。提示LOX-1与mi R-98可能存在密切联系。将mi R-98 mimic和inhibitor转染入细胞,发现LOX-1表达被明显抑制或增强。由于诸多因素参与AS的形成,本研究同时检测了氧化应激反应(活性氧自由基[reactive oxygen species,ROS]),内皮功能相关因子表达(内皮素-1[endothelin-1,ET-1]、内皮型一氧化氮合酶[endothelial nitric oxide synthase,e NOS]、诱导型一氧化氮合酶[inducible nitric oxide synthase,i NOS]),内皮细胞通透性,泡沫细胞形成(巨噬细胞油红O染色)。结果发现,与对照组相比,mi R-98 mimic转染的内皮细胞组ROS、ET-1、i NOS表达降低,e NOS表达增高,泡沫细胞形成减少。mi R-98inhibitor作用与之相反。提示mi R-98可能参与调控AS的形成。3.mi R-98对小鼠AS的影响为模拟AS的体内环境,本研究采用LDLr-/-小鼠模型,并对其注射agomi R-98或antagomi R-98,检测小鼠主动脉LOX-1、ET-1、e NOS、i NOS表达情况,以及其对内皮通透性以及脂质沉积的影响。结果发现,agomi R-98可明显抑制LOX-1、ET-1、i NOS的表达,增加e NOS表达,减少动脉内膜的脂质沉积,并降低内皮通透性改善内皮功能。而antagomi R-98的作用与之相反。进一步提示mi R-98可能参与调控AS的形成。4.验证LOX-1是mi R-98的靶基因为明确LOX-1是否为mi R-98的靶基因,本研究采用荧光素酶报告基因进行检测,结果发现mi R-98 mimic组的荧光强度较对照组明显降低,而相同浓度的mi R-98 inhibitor则明显增高;当点突变LOX-1 3’-UTR后(沉默mi R-98与LOX-1的结合位点),其已被mi R-98 mimic抑制的荧光强度又恢复正常。提示LOX-1是mi R-98的靶基因,其结合位点是LOX-1 3’-UTR上的447-453片段。由此验证了假设并得出结论:mi R-98通过抑制LOX-1表达抑制AS的形成。5.小结miR-98通过抑制其靶基因LOX-1参与调控AS早期的特征性病理变化过程,抑制活性氧自由基生成,抑制ET-1、i NOS,增加e NOS的表达,从而改善动脉内皮功能,以及抑制泡沫细胞形成和动脉内膜脂质沉积。本研究为应用微小RNA预防和治疗AS提供了理论和实验依据。
[Abstract]:Atherosclerosis (AS) is the most important cause of cardiovascular disease. It participates in a variety of fatal cardiovascular diseases such as myocardial ischemia, angina, myocardial infarction, and heart failure. How to inhibit the formation of AS and reduce the incidence and mortality of AS cardiovascular disease has become the goal of the joint effort in this field,.AS The formation mechanism is complex, including the following factors: oxidative stress, arterial endothelial injury and dysfunction, foam cell formation, and subsequent lipid deposition and thrombosis. The oxidative stress, endothelial dysfunction and foam cell formation are the process of characteristic venereal disease in the early AS. Micro RNAs (MI Rs) It is a small RNA fragment of about 22 nucleotides that effectively inhibits the transcription and translation of M RNA by binding to the messenger RNA 3 'end of the target gene to exert different biological effects. Studies have shown that MI Rs is involved in a variety of cardiovascular diseases, such as myocardial ischemia, myocardial fibrosis, hypertension, diabetic cardiomyopathy, heart failure and AS. However, these studies are still in the early stages. How mi Rs regulates the mechanism of AS and how to use mi Rs to treat AS is a pending problem. The previous study found that some mi Rs in the plasma of AS patients had a significant difference compared with those of healthy volunteers. Through bioinformatics, the microarray data were predicted and analyzed, and these mi Rs were found. There is a common target gene, the oxidized low density lipoprotein receptor -1 (lectin-like oxidized low-density lipoprotein receptor-1, LOX-1).LOX-1 is a receptor on the cell membrane of oxidized low density lipoprotein (oxidized low density lipoprotein), and has the characteristics of the scavenger receptor. Irritable, endothelial dysfunction, foam cell formation and lipid deposition. Therefore, in this study, a scientific selection method is used to select a most representative mi R to study its effect on AS and to further explore its possible mechanisms. The main contents of this study include the following four parts: 1.AS patients and healthy volunteers, plasma Mi R Microarray technique was used to detect the expression of MI Rs and m RNAs in AS patients and healthy volunteers. It was found that the expression of 28 mi Rs and 32 m RNA showed significant changes. The binding site.LOX-1 is an important regulatory factor in the formation of AS, suggesting that MI Rs may affect the formation of AS by regulating LOX-1 expression in.2. in vitro simulated AS environment and evaluating the effect of MI R-98 on AS formation in vitro. /m L), the expression of MI R-98 gradually decreased, and at the same time, the expression of LOX-1 increased gradually. It suggested that LOX-1 and MI R-98 may have a close relationship. Free radical [reactive oxygen species, ROS]), expression of endothelial function related factors (endothelin -1[endothelin-1, ET-1], [endothelial nitric oxide synthase, e NOS], inducible nitric oxide synthase), endothelial cell permeability, foam cell formation (macrophage oil red) The results showed that, compared with the control group, the expression of ROS, ET-1, I NOS in the MI R-98 mimic transfected endothelial cells decreased, the expression of E NOS increased, and the formation of.Mi R-98inhibitor was opposite to that of the foam cell formation. The expression of LOX-1, ET-1, e NOS and I NOS in mouse aorta, as well as the effect on endothelial permeability and lipid deposition were detected by r-/- mouse model, and the effect of agomi R-98 or antagomi R-98 on the expression of LOX-1, ET-1, e NOS and I NOS. The skin permeability improves the endothelial function, and the effect of antagomi R-98 is contrary to it. Further hints that MI R-98 may participate in the regulation of AS formation by.4. verification that LOX-1 is the target of MI R-98 because LOX-1 is the target gene of MI R-98, and the fluorescence intensity of the group is detected by the luciferase reporter gene. The control group was significantly lower, while the same concentration of MI R-98 inhibitor was significantly higher; when the point mutation LOX-1 3 '-UTR (the binding site of silent mi R-98 and LOX-1), the fluorescence intensity that had been suppressed by Mi R-98 mimic was restored to normal. It is assumed that MI R-98 inhibits the formation of AS by inhibiting the expression of LOX-1 and inhibits its target gene LOX-1 by inhibiting the target gene LOX-1 to regulate the characteristic pathological changes of the early AS, inhibiting the formation of reactive oxygen radicals, inhibiting the ET-1, I NOS, and increasing the expression of E, thus improving the function of the artery endothelium and inhibiting the formation of foam cells. This study provides a theoretical and experimental basis for the prevention and treatment of AS with tiny RNA.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R543.5

【相似文献】