Exenatide对高糖高脂诱导的NIT-1细胞损伤和炎症因子过表达的影响

发布时间：2018-03-02 12:23

本文选题：艾塞那肽　切入点：高糖高脂　出处：《四川医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景:糖尿病大多伴有糖脂代谢紊乱,而长期的高糖、高脂会诱导细胞因子的大量生成,引起胰岛β细胞的炎症反应,造成胰岛β细胞的损伤。但糖脂毒性诱导β细胞功能障碍的具体机制目前尚不十分清楚。糖尿病也是自身免疫和慢性炎症性疾病,主要表现为慢性、低度、持续的炎性标志物水平的升高。炎症介质既是糖尿病的病理标志物,也是糖尿病及其并发症发生发展的原因。大量研究表明TNF-α、IL-1β、IL-6和CRP是预测糖尿病慢性炎症反应的敏感性标志物。Toll样受体(Toll-like receptors,TLRs)信号通路是免疫炎症反应重要的信号通路,其信号的过度激活能直接诱导机体自身免疫的破坏和耐受丢失,启动或者加速自身免疫性疾病进程;因此,TLR信号通路可能参与和影响糖尿病及其慢性并发症的发生和发展。研究证实胰高血糖素样肽-1(Glucagon-like peptide-1,GLP-1)受体激动剂能够抑制动脉粥样硬化和抗氧化应激,改善血管内皮功能,具有降低心血管事件风险的潜在益处,而动脉粥样硬化本身也是一种动脉壁的慢性炎症,抗粥样硬化的作用很大程度上依赖于抗炎效应的发挥;这些研究提示艾塞那肽(Exenatide)保护胰岛β细胞的作用可能与抗炎抗免疫作用有关,除降糖以外,还可能减少糖尿病患者体内氧化应激和炎症反应。目的:观察高糖高脂与TLR3的关系,了解糖脂毒性诱导免疫炎症反应及胰岛β细胞损伤可能的机制。进一步观察高糖高脂环境下,艾塞那肽与TLR3的关系,了解艾塞那肽是否具有抗炎作用,是否能够逆转糖脂毒性对胰岛β细胞的损伤。方法:培养胰岛β细胞株(NIT-1),经高糖高脂刺激后,进行艾塞那肽干预,应用CCK-8法观察细胞增殖情况,应用实时荧光定量PCR(realtime PCR)检测TLR3 mRNA表达,酶联免疫吸附法(ELISA)检测细胞培养上清液中TNF-α、IL-1β、IL-6、CRP、补体C3及补体C4的表达,放射免疫法检测葡萄糖刺激后胰岛素分泌情况。结果:高糖高脂刺激后,TLR3 mRNA表达显著上调,TLR3固有免疫炎症信号通路激活,产生大量免疫炎症因子,TNF-α、IL-1β、IL-6、CRP、补体C3及补体C4的表达显著增加,胰岛β细胞增殖及胰岛素分泌能力受到显著抑制;并且高糖高脂协同效应大于单独的糖脂毒性作用。给予不同浓度艾塞那肽干预后,TLR3mRNA显著下降,TNF-α、IL-1β、IL-6、CRP、补体C3及补体C4表达减少,胰岛β细胞增殖率及胰岛素分泌水平增加,呈现浓度依赖性。结论:高糖高脂能显著上调胰岛β细胞TLR3mRNA的表达,激活TLR3固有免疫炎症信号通路,产生大量免疫炎症因子,导致胰岛β细胞生长活力下降,细胞增殖及胰岛素分泌能力受到抑制;并且高糖高脂协同效应大于单独的糖脂毒性作用。TLR3可能在糖脂毒性诱导的胰岛β细胞损伤过程中扮演着重要角色。高糖高脂环境下,艾塞那肽能够显著下调TLR3 mRNA的表达,TLR3免疫炎症信号通路的激活受到抑制,免疫和炎症因子表达显著下调,并且具有浓度依赖性;β细胞增殖及胰岛素分泌水平增加。艾塞那肽可能通过抑制TLR3免疫炎症信号通路,产生抗炎效应,对抗糖脂毒性对胰岛β细胞的损伤。
[Abstract]:Background: diabetes mostly associated with lipid metabolic disorders, and long-term high sugar, high fat will generate large induced by cytokines, inflammation of pancreatic beta cells, resulting in islet beta cell injury. But the specific mechanism of glucolipotoxicity induced beta cell dysfunction is currently unclear. Diabetes is autoimmune and chronic inflammatory diseases, the main manifestation of chronic, low-grade, persistent inflammatory markers increased. Pathological markers of inflammatory mediators is also the reason of the development of diabetes, diabetes and its complications occurred. Many studies showed that TNF- alpha, IL-1 beta, IL-6 and CRP is a sensitive marker of.Toll like receptors in predicting diabetes chronic inflammation (Toll-like receptors, TLRs) signaling pathway is an important immune inflammatory pathway, damage tolerance and excessive activation of its signal can directly induce autoimmune lost Lost, starting or accelerating autoimmune disease process; therefore, the occurrence and development of TLR signaling pathway may be involved in and the effects of diabetes and its chronic complications. Studies have confirmed that glucagon like peptide -1 (Glucagon-like peptide-1 GLP-1) receptor agonist can inhibit atherosclerosis and oxidative stress, improve endothelial function, with potential benefits to reduce the risk of cardiovascular events, and atherosclerosis itself is a kind of chronic inflammation of the arterial wall, anti atherosclerotic effect depends largely on anti-inflammatory effect; these studies suggest that exenatide (Exenatide) protects the islet beta cells may be associated with anti-inflammatory and anti immune function, in addition to hypoglycemic outside, may also reduce oxidative stress in patients with diabetes and inflammatory reaction. Objective: To observe the relationship between the high fat and high sugar and TLR3, understand glucolipotoxicity induced immune inflammation The mechanism may be and islet cell injury. To observe the effect of high glucose and lysophosphatidylcholine, relationship between exenatide and TLR3, to understand whether exenatide has anti-inflammatory effect, can damage reversal glucolipotoxicity on pancreatic islet cells. Methods: cell culture (NIT-1), the high fat and high sugar after stimulation of exenatide intervention, CCK-8 method was used to observe cell proliferation and application of real-time fluorescence quantitative PCR (realtime PCR) to detect the expression of TLR3 mRNA and enzyme-linked immunosorbent assay (ELISA) detection of cell culture supernatant of TNF- alpha, IL-1 beta, IL-6, CRP, expression of complement C3 and complement C4 in the situation detection of glucose stimulated insulin secretion by radioimmunoassay. Results: high fat and high glucose stimulation, TLR3 mRNA expression was significantly up-regulated in TLR3 innate immune inflammatory signaling pathway, resulting in a large number of inflammatory factors, TNF- alpha, IL-1 beta, IL-6, CRP, expression of complement C3 and complement C4 Increased proliferation and insulin secretion ability was significantly inhibited; and high glucose and fat synergistic effect is greater than the glycolipid toxicity alone. Treated with different doses of exenatide intervention, TLR3mRNA decreased significantly, TNF- alpha, IL-1 beta, IL-6, CRP, complement C3 and complement C4 decreased expression of islet beta cells the proliferation rate and insulin secretion increased in a dose-dependent manner. Conclusion: high glucose and fat can up regulate the expression of TLR3mRNA in islet beta cells, activation of TLR3 signaling pathway in innate immune inflammation, resulting in a large number of inflammatory factors, leading to pancreatic islet beta cell growth activity decreased cell proliferation and insulin secretion was inhibited and high glucose and fat; coordination plays an important role in islet beta cell damage effect is greater than the glycolipid toxicity of.TLR3 alone may be induced in glucolipotoxicity. The high fat and high glucose environment, exenatide can TLR3 mRNA expression was significantly reduced, the activation of TLR3 signaling pathway inhibited immune inflammation, immune and inflammatory factor expression were significantly reduced, and in a concentration dependent manner; beta cell proliferation and insulin secretion increased. Exenatide may inhibit the TLR3 inflammatory signaling pathway, produce anti-inflammatory effect, damage against glucolipotoxicity on beta cells.

【学位授予单位】：四川医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

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