【作者】 周宁；

【导师】 厉有名；

【作者基本信息】 浙江大学 ， 内科学， 2013， 博士

【摘要】 研究背景与目的酒精性肝病(alcoholic liver disease, ALD)是长期过量饮酒所致的肝损伤,包括酒精性脂肪肝、酒精性肝炎、酒精性肝硬化三种主要病理类型。随着人们饮酒量的增加,ALD的发病率有逐年增长趋势。2011年美国男性和女性ALD的患病率分别为7.4%、4.0%。ALD在俄国的发生率约为5%。我国虽然缺乏大规模的流行病学数据,但据统计,ALD已成为仅次于病毒性肝炎的第二大肝病。2000年浙江省ALD流行病学调查发现,人群患病率为4.34%。ALD是一种遗传-行为-环境相互作用的慢性疾病,其发病机制非常复杂,包括乙醇及其衍生物的代谢、营养失衡、免疫因素、氧化应激、内质网应激、缺氧、炎症介质、自噬与凋亡、基因多态性、microRNA和表观遗传学等。但这些诠释尚未涵盖所有的发病机制,ALD发生发展过程中复杂的分子事件直接导致了各种针对ALD的治疗措施收效不显。因此,深入研究其发病机制对指导本病的防治和提高患者的生活质量意义深远。本实验在成功构建ALD动物模型的基础上,分析了ALD发生发展过程中肝脏基因和蛋白质表达谱的动态变化,以期为认识ALD发生发展机制提供新线索。研究方法首先,用酒精给大鼠连续灌胃1月和3月,观察肝脏病理变化。其次,采用基因芯片技术,对酒精性脂肪肝组、酒精性肝炎组和正常对照组大鼠肝脏基因表达变化进行两两比较分析,并应用分子注释系统(MAS)进行基因本体(GO)功能注释和Pathway (KEGG和GenMAPP)分析。第三,应用双向荧光差异凝胶电泳(2D DIGE)技术分析不同病理状态的模型组和对照组大鼠肝脏蛋白质表达谱的差异,所得到的差异蛋白质点经质谱仪(MALDI-TOF/TOF)进行检测以鉴定差异蛋白,并结合生物信息学手段对差异蛋白进行功能分析和相互作用网络分析。结果动物实验观察到了肝细胞脂肪变性、炎症细胞浸润、坏死,与人类酒精性脂肪肝和酒精性肝炎的病理表现相似。基因组学研究方面,分别筛选出显著差异表达的基因285个(酒精性脂肪肝组与正常对照组比较)、318个(酒精性肝炎组和正常对照组比较)。两种病理状态的大鼠在肝脏基因的表达上无显著的统计学差异。这些差异基因主要参与了氧化还原、各种代谢(包括脂质、乙酰辅酶A、p-内酰胺类抗生素、谷氨酸盐、氨基酸、碳水化合物)、细胞分化及凋亡、补体激活等200多种生理过程和分子功能,以及近百条信号通路。通过分析及比较,发现了一些具有明显阶段特异性的差异基因,它们分别参与了不同的代谢途径。H2-T3,Aldhlll,Pklr等基因在酒精性脂肪肝阶段上调；同阶段下调的基因有：Pla2g12a, Hes1,Gcnt2,Adh4等。Coq6, Idi1,Fdps, Ppox, Gck, Npas2,Ddhdl等基因在酒精性肝炎阶段上调；同阶段下调的基因有:C7,Alas2, Got1,Aoc3,Vcam1,Mmp2, G6pc等。这些基因可能因作用于不同的代谢途径而导致了不同的病理改变。其中,大部分基因罕见报道。值得关注的是,Pla2gl2a基因参与了VEGF, MAPK等多条信号通路,而MAPK信号通路又通过细胞色素P4504a8基因与PPAR信号通路有着联系。Pla2g12a基因如何受VEGF信号转导通路的调控,其引起酒精性脂肪肝发生的机制尚待深入研究。此外,差异基因相互作用网络图分析发现了位于网络中心的关键节点基因周期素D1(Ccndl).我们还从参与ALD发病机制的信号通路中选择了PPAR.胰岛素、MAPK.粘着斑和P53这5条信号通路,并挑选这些通路中的37个基因进行了荧光定量PCR验证,验证结果与芯片检测结果一致。其中,脂肪酸合成酶基因(Fasn)和肉毒碱棕榈酰基转移酶1A基因(Cpt1a)的表达量随ALD病程的进展分别持续增强和减弱。蛋白组学研究方面,共鉴定了49个差异蛋白。除去冗余蛋白,酒精性脂肪肝和酒精性肝炎两个病理阶段的差异蛋白共35个。它们分别与三大物质的代谢、尿素合成、脂肪酸β氧化、细胞骨架、免疫防御、电子转移、细胞交通、ATP结合、抗氧化等功能有关。其中,8个蛋白的表达水平在酒精性脂肪肝和酒精性肝炎阶段均明显上调,分别是：碳酸酐酶3、谷胱甘肽转移酶Mu1和Mu2、衰老标记蛋白、电子转移黄素蛋白亚单位α、腺苷高半胱氨酸酶、类甘油醛-3-磷酸脱氢酶和过氧化氢酶(catalase, CAT)。而ATP合成酶亚单位d和β肌动蛋白FE-3的表达水平在这两阶段均明显下调。提示这些蛋白可能与ALD的发生发展有关。尤其是CAT,在应用Genomatix软件分析差异蛋白的相互作用网络后,发现它处于网络的中心,故推测这些差异蛋白可能因为影响了CAT而最终导致了ALD。综合文献报道,我们认为衰老标记蛋白可以作为诊断ALD的生物学标志物,它可能系通过胰岛素代谢途径而影响了ALD的发生发展。另一发现是,谷胱甘肽转移酶Mul在ALD发展过程中的表达量稳步上升,间接反映了ALD发生发展过程中氧化应激水平的不断加重。结论1成功地构建了ALD大鼠模型；2发现了一些阶段特异性的代谢途径和差异基因,其中Pla2g12a基因及其参与的VEGF信号途径在酒精性脂肪肝发病中的作用值得关注。与ALD病理严重程度一致的Fasn和Cpt1a基因,以及Ccndl节点基因对ALD分子机制的研究、早期诊断及分子靶向治疗研究有较好的提示作用。3鉴定到了10个与ALD的发生发展过程密切相关的蛋白。其中,RGN可以作为诊断ALD的生物学标志物；GSTM1有望成为判断ALD严重程度的生物学指标；CAT代谢途径在ALD发生发展中的作用不容忽视。更多还原

【Abstract】 Background/aimsAlcoholic liver disease (ALD) is a major healthcare problem worldwide which results from consumption of large amount of alcohol during a sustained period of time in a subset of alcoholics. Globally, in2010, alcohol-attributable liver cirrhosis was responsible for493,300deaths [5] The pathological spectrum runs the gamut from alcoholic fatty liver (AFL) to alcoholic hepatitis (AH), fibrosis, and frank cirrhosis. ALD affects approximately90%to100%of long-term heavy drinkers. Once alcoholic hepatitis develops, abstinence from drinking reverses this condition in only10%of the individuals. About15-40%of the patients rapidly progress towards the end-stage liver disease, digestive hemorrhage and superimposed hepatocellular carcinoma (HCC). Epidemiology in2011showed the prevalence of ALD in US was7.4%in men and4%m women.ALD results from a complex interaction between behavioral, environmental, and genetic factors. The molecular pathogenesis of ALD involves alcohol metabolism and secondary mechanisms, including oxidative stress, endotoxin, cytokines, immune regulators, ischaemia, metabolic disturbances (alteration of methionine metabolism, and so on), nutritional factors, and genetic predisposition. Despite decades of intensive investigation, the basic pathophysiological mechanisms responsible for ALD have remained elusive. Hence, there is a great need for using new tools to comprehend the mechanistic perspectives underlying the complex pathogenesis of ALD. Moreover, it is vital to identify non-invasive biomarkers that can accurately distinguish the different ranges of chronic alcoholic liver injuries and therapy thereof.This dissertation consists of three parts:establishment of ALD rat model, gene expression analysis by microarray, and proteomic analysis of the spectrum of ALD.MethodsTo mimic the progress of human ALD, rats were treated with ethanol by means of intragastric infusion for1and3month respectively. After the animals were killed, liver histology and total RNA analysis by microarray were performed. Then differential expressed genes were annotated with GO and Pathway analysis. Real time-PCR was used for further validation. Changes in protein expression of liver samples from each of the three groups of subjects (controls, AFL, and AH) were analyzed by2D DIGE combined with MALDI-TOF/TOF analysis.ResultsIn the different experimental groups,285(AFL vs. Con) and318(AH vs. Con) different genes were differentially expressed. Of which, there are137/142up-regulated genes, and148/176down-regulated ones in AFL group and AH group respectively as compared to the control group. The majority of the trafficking genes are involved in the degradation and synthesis of fatty acid, steroid biosynthesis, inflammatory response, oxidative stress, pentose phosphate pathway, nuclear receptors, and proteasome degradation. The expressions of37genes in5pathways done by real time quantitative PCR had coincidence with that of microarray. Dozens of phase specific genes were identified, which may play some roles in the development of ALD. Of them, phospholipase A2group XIIA gene (Pla2g12a) and the signaling pathway of vascular endothelial growth factor (VEGF) deserve mention. Notably, Ccndl was found to be a node gene in the gene network map. It was activated or modulated by other genes. Additionally, fatty acid synthase (Fasn) gene kept rising and carnitine palmitoyltransferase1a (Cpt1a) gene kept down during the process of ALD.Forty-nine proteins exhibited significant changes (ratio≥1.5or≤-1.5, P＜0.05). Of which,45(AFL vs. Con) and29(AH vs. Con) proteins were identified, with part of them identical. Thirty-five non-redundant proteins were determined to be involved in numerous biological functions, including metabolism of amino acid, lipid and carbohydrate. Catalase was a node protein in the network map after analyzing by Genomatix software. It had extensive connection with other proteins, which means these proteins might have some roles in ALD by involving in ethanol-catalase pathway. Besides, we found that the expression levels of8proteins were significantly up-regulated (>1.5-fold) in alcohol fed group as compared with the control one, namely catalase, carbonic anhydrase3,glutathione S-transferase Mul (GSTM1), glutathione S-transferase Mu2, regucalcin (RGN), electron transfer flavoprotein subunit alpha, adenosylhomocysteinase, and glyceraldehyde-3-phosphate dehydrogenase-like. Meanwhile, the expression levels of ATP synthase subunit d and beta actin FE-3were significantly down-regulated (<1.5-fold) in alcohol fed group as compared with the control one. Interestingly, regucalcin was reported to have close relationship with insulin resistance and could be used as a biomarker of ALD. Our research testified it. We infer that regucalcin may promote the process of ALD by impairing hepatic insulin signaling. Moreover, GSTM1was steadily up-regulated in the rat liver with the progression of ALD.Collectively, the results of this study provide our knowledge to the pathopoiesis of ALD by identifying the intrahepatic gene and protein expression profiles of rats. In addition, some ALD-related genes and proteins were revealed, which are plausible of biologic markers of the disease, such as Fasn, Cptla, Ccndl gene, RGN and GSTM1. The relationship between Pla2gl2a in VEGF signaling pathway and ALD should be investigated further. Likewise, ethanol-catalase pathway cannot be ignorant. But the results should be confirmed by further studies.更多还原