晚期氧化蛋白产物诱导大鼠痛觉过敏及其机制的研究

发布时间：2018-03-12 09:56

本文选题：晚期氧化蛋白产物　切入点：痛觉过敏　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：痛觉过敏是一种由神经系统受损或者功能障碍引起的机体对疼痛刺激的感觉阈值下降,轻微刺激便引起疼痛感觉的临床常见症状。痛觉过敏明显降低患者的生活质量,带给患者乃至整个社会巨大的经济和精神负担。痛觉过敏的发病机制十分复杂,其中最主要的致病因素就是体内氧化应激反应失代偿后产生的活性氧自由基(Reactive Oxygen Species,ROS)。晚期氧化蛋白产物(Advanced Oxidation Protein Products,AOPPs)在体内的最主要来源是血浆白蛋白与氧化应激所形成的含氯氧化物反应而成,是一类含有二酪氨酸的蛋白产物。AOPPs不仅被认为是氧化应激导致蛋白质损伤的标记物,同时也是引起氧化应激和炎症反应的重要介质。目前尚无相关研究表明AOPPs是否参与痛觉过敏病理生理过程。本研究旨在观察AOPPs是否会诱导SD大鼠出现痛觉过敏并探究其中的分子机制。为了验证AOPPs参与SD大鼠痛觉过敏模型,本研究利用经典的完全弗氏佐剂(Complete Freund' sAdjuvant,CFA)诱导SD大鼠痛觉过敏动物模型,通过氯胺-T法检测SD大鼠血浆中AOPPs的含量为对照组SD大鼠的1.6倍。此外,给予正常SD大鼠静脉注射体外配制的AOPPs溶液,利用电子Von Frey测痛仪检测SD大鼠机械缩足阈值明显下降。对AOPPs诱导痛觉过敏的SD大鼠背根神经节(Dorsal Root Ganglia,DRG)组织进行免疫荧光染色和蛋白免疫印迹检测,结果表明AOPPs明显上调组织中NADPH氧化酶1(Nox1)、NADPH氧化酶 4(Nox4)、瞬时感受器电位(Transient Receptor Potential,TRP)Vanilloid 1 通道(TRPV1)和降钙素基因相关肽(Calcitonin Gene-Related Peptide,CGRP)的表达。将原代培养的背根神经节神经元细胞作为体外研究的对象,利用DCFH-DA荧光染料通过多功能酶标仪和激光共聚焦扫描显微镜检测发现AOPPs诱导DRG神经元细胞产生大量的ROS,并且通过小干扰RNA技术,发现细胞内大量的ROS来自于AOPPs对细胞中Noxl蛋白和Nox4蛋白的激活。除此之外,利用细胞蛋白免疫印迹的方法发现AOPPs可以明显激活TRPV1离子通道,并且促进DRG神经元细胞合成和释放大量神经肽类物质CGRP,同时引起钙离子大量流入DRG神经元细胞质中,扰乱细胞的稳态。综上所述,本研究发现AOPPs在CFA诱导痛觉过敏的大鼠血浆中明显升高,并且成功地建立了体外配制的AOPPs诱导大鼠痛觉过敏动物模型。通过一系列体内外实验,明确了 AOPPs通过激活Noxl和Nox4蛋白产生大量的ROS,进一步激活TRPV1离子通道,从而导致DRG神经元细胞分泌CGRP和大量钙离子流入细胞质内,最终导致痛觉过敏发生。
[Abstract]:Hyperalgesia is a common clinical symptom of pain stimulation caused by nervous system damage or dysfunction, which is caused by a decrease in the body's threshold for pain stimulation. Hyperalgesia can significantly reduce the quality of life of patients. Bring patients and even the whole society a huge economic and mental burden. The pathogenesis of hyperalgesia is very complicated. The main pathogenic factor is reactive Oxygen species produced by oxidative stress decompensation in vivo. The main source of advanced Oxidation Protein products AOPPs in vivo is plasma albumin and oxidative stress. Formed by the reaction of chlorinated oxides, AOPPs are a class of protein products containing dityrosine. AOPPs are not only considered to be markers of protein damage caused by oxidative stress. At the same time, it is also an important medium to cause oxidative stress and inflammation. There is no relevant study on whether AOPPs is involved in the pathophysiological process of hyperalgesia. This study was designed to observe whether AOPPs could induce hyperalgesia in SD rats. To investigate the molecular mechanisms involved, AOPPs was involved in a hyperalgesia model in SD rats. In this study, the hyperalgesia animal model of SD rats was induced by the classical complete Freundte Freundte's Adjuvant CFA. the plasma AOPPs content in SD rats was measured by chloramine-T method, which was 1.6 times as high as that in the control group. Normal SD rats were given intravenous injection of AOPPs solution prepared in vitro, The mechanical foot contraction threshold of SD rats was detected by electronic Von Frey. The tissue of dorsal root ganglion of SD rats induced by AOPPs was detected by immunofluorescence staining and Western blotting. The results showed that AOPPs upregulated the expression of NADPH oxidase 1 (Nox1), transient Receptor potential (TRP) and calcitonin Gene-Related Peptidee (CGRP1) in tissues. The primary cultured neurons of dorsal root ganglion (DRG) were used as primary cultured neurons of dorsal root ganglion (DRG). Object of study in vitro, DCFH-DA fluorescence dye was used to detect ros in DRG neurons by multifunctional enzyme labeling instrument and confocal laser scanning microscope. AOPPs induced a large number of ROSs in DRG neurons, and small interference RNA technique was used to detect ROSs in DRG neurons. It was found that a large amount of ROS in cells was derived from the activation of Noxl protein and Nox4 protein by AOPPs. In addition, we found that AOPPs could activate TRPV1 ion channel by cell Western blot. It also promotes the synthesis and release of a large number of neuropeptides from DRG neuron cells, and causes calcium ions to flow into the cytoplasm of DRG neurons in large quantities, thus disrupting the steady-state of the cells. In this study, we found that AOPPs increased significantly in the plasma of CFA induced hyperalgesia rats, and successfully established an in vitro AOPPs induced hyperalgesia animal model in rats. It is clear that AOPPs activates a large number of ros by activating Noxl and Nox4 proteins, and further activates the TRPV1 ion channel, which leads to the release of CGRP and a large number of calcium ions into the cytoplasm by DRG neurons, leading to hyperalgesia.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R402

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