橙皮苷对斑马鱼酒精性脂肪肝的治疗作用及其机制研究

发布时间：2018-03-05 07:08

  本文选题：斑马鱼　切入点：橙皮苷　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景及目的酒精性脂肪肝(alcoholic fatty liver disease,AFLD)以长期大量饮酒而导致脂肪在肝细胞中过度贮积为主要特征,是酒精性肝病(alcoholic liver disease,ALD)的早期阶段,及时治疗可以得到有效控制甚至逆转,对ALD的防治意义重大。中医药治疗AFLD历史悠久且疗效显著,深入研究中药及其成分对AFLD的作用效果及机制,对AFLD的防治意义重大。橙皮苷是中药陈皮、枳实的主要成分,具有抗炎、抗氧化、降胆固醇等多种活性作用,对AFLD有一定的疗效,而具体的作用机制尚不明确。斑马鱼因其独特的生物学优势,现已广泛应用于疾病模型的建立和药物筛选,其中斑马鱼急性酒精性脂肪肝模型的建立,为进一步研究AFLD的发病机制及开展药物研究开辟了新的途径。基于以上背景,本研究利用斑马鱼急性酒精性脂肪肝模型,旨在进一步研究橙皮苷对AFLD的保护作用及其机制,为中药在AFLD上的应用提供依据。研究方法1.实验动物AB野生型斑马鱼、肝脏荧光转基因斑马鱼Tg(1fabp1Oα:eGFP)。2.酒精和药物处理随机挑选4dpf(days post ferti1ization)的斑马鱼于培养皿中,对照组只加培养用水,模型组中加入含350mM乙醇的培养用水,培育32小时,建立急性酒精性脂肪肝模型,收集一部分样品进行后续研究。随后,随机将模型组分为5组:0.1%DMSO 组、模型组、3 个浓度橙皮苷处理组(25ug/mL、12.5ug/mL、6.25ug/mL),与对照组一起置于28.5℃恒温箱培养中培养48小时,收集各组幼鱼进行下一步研究。3.试验技术通过油红O染色、尼罗红染色、HE染色技术观察斑马鱼肝脏形态学变化、脂滴沉积情况及组织病理学改变;运用qPCR技术来检测目标基因的mRNA表达。4.数据统计:采用spss20软件进行数据分析,计量资料以均数±标准差(x±s)表示,P0.05视为差异有统计学意义。实验结果1.相比对照组,置于含350mM乙醇的水中培育32小时的斑马鱼肝组织形态改变和脂肪变程度更明显,证实成功建立斑马鱼急性酒精性脂肪肝模型。2.油红O染色、尼罗红染色显示橙皮苷可显著减轻酒精导致的肝内脂滴沉积和肝脏形态学改变,斑马鱼石蜡切片的HE染色证实橙皮苷能改善酒精导致的肝脏病理变化。说明橙皮苷能改善酒精所致的斑马鱼肝脏脂肪变。3.橙皮苷通过调节酒精代谢、脂质代谢以及抵抗内质网应激和DNA损伤来抵御酒精对肝脏的损害,具体表现为下调cyp2y3,cyp3a65,fads2,fasn,hmgcra,hmgcrb,chop,gadd45αa,edem1基因的mRNA表达量。
[Abstract]:Background and objective\\\. It is of great significance to prevent and cure ALD. Chinese medicine has a long history and remarkable curative effect in the treatment of AFLD. It is very important to study the effect and mechanism of Chinese medicine and its components on AFLD. Hesperidin is the main component of Fructus Aurantii and Fructus Aurantii. Zebrafish have been widely used in the establishment of disease models and drug screening because of their unique biological advantages. The establishment of acute alcoholic fatty liver model of zebrafish opens up a new way to further study the pathogenesis of AFLD and the development of drug research. Based on the above background, the model of acute alcoholic fatty liver of zebrafish was used in this study. The aim of this study was to further study the protective effect of hesperidin on AFLD and its mechanism, and to provide the basis for the application of traditional Chinese medicine in AFLD. Liver fluorescence transgenic zebrafish Tg(1fabp1O 伪: eGFP.2.Alcohol and drug treatment were randomly selected from zebrafish (4dpffdays post fertigation) in petri dish. The control group was treated with culture water only, and the model group was treated with 350mM ethanol for 32 hours. An acute alcoholic fatty liver model was established and some samples were collected for follow-up study. Then the model group was randomly divided into 5 groups: 0.1 DMSO group. The model group with 3 concentrations of hesperidin was cultured in 28.5 鈩，

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