【作者】 张雪群；

【导师】 厉有名；

【作者基本信息】 浙江大学 ， 内科学， 2008， 博士

【摘要】 近年来随着人们生活方式和饮食结构的改变,非酒精性脂肪性肝病(Nonalcoholic fatty liver disease,NAFLD)的患病率日益增高,成为导致慢性肝病的重要原因之一,构成日益严重的社会健康问题。NAFLD包括一系列相互联系的病理改变,从单纯性脂肪肝、脂肪性肝炎,到肝纤维化和肝硬化,甚至可以进展为肝细胞癌。目前认为,NAFLD与肥胖、糖尿病、高脂血症等代谢综合征密切相关,而高脂饮食是引起肥胖及其相关并存症高发的重要原因。摄入含过多饱和脂肪的食物可以直接导致肝细胞的脂肪浸润及氧化损伤,但其确切的分子机制尚未明确。用高脂饲料喂养SD大鼠可以成功复制不同病变程度的NAFLD动物模型。本研究采用蛋白质组学的技术手段,系统分析了高脂饮食相关的NAFLD发生发展过程中肝脏蛋白质表达谱的动态变化,发掘了一批与NAFLD疾病进程密切相关的功能蛋白质群,结合生物信息学手段系统分析差异蛋白的功能和调控网络,从而为深入探讨NAFLD发生发展的分子机制奠定了基础。用高脂饲料喂养SD大鼠4周、12周和24周,肝脏病理分别表现为肝细胞脂肪变性、脂肪变性伴炎症细胞浸润,以及脂肪变性伴炎症细胞浸润及轻度纤维化,分别与NAFLD的单纯性脂肪肝(SS),脂肪肝合并非特异性炎症(NSI)及脂肪性肝炎(NASH)这三个阶段的病理表现相似。随后,我们利用差异凝胶电泳技术(DIGE)分析了不同造模时间的正常组和模型组大鼠肝脏蛋白质表达谱的差异,所得到的差异蛋白质点用MALDI-TOF/TOF质谱仪进行检测,共鉴定了95个差异蛋白,其中SS阶段的差异蛋白53个,NSI阶段的差异蛋白42个,NASH阶段的差异蛋白49个。这些差异蛋白在功能上具有明显的阶段特异性。其中参与脂肪酸氧化的酶在SS阶段明显下调,可能是造成该阶段肝细胞脂肪变性的重要原因;多个参与线粒体呼吸链和氧化磷酸化的酶在NSI阶段表达下调,提示线粒体功能障碍在这一阶段的重要作用;最后在NASH阶段,多个细胞骨架蛋白的表达都明显上调,很可能与该阶段肝细胞的结构和功能紊乱相关。进一步对这些差异蛋白进行相互作用网络的分析发现,多个差异蛋白的表达受到PPARα和C/EBPα这两个转录因子的调控,随后我们通过Western blot方法检测到PPARα在疾病的三个阶段都明显下调,而C/EBPα在SS和NSI阶段明显上调,提示这两个转录因子在NAFLD的发生发展中发挥了重要作用。此外,我们还发现4个内质网分子伴侣(PDI,PDIA3,GRP78和ERp29)在进展期NSI和NASH阶段明显上调。内质网分子伴侣表达上调是内质网应激的一个重要特点,资料显示内质网应激在肥胖、2型糖尿病等代谢性疾病中均发挥了重要作用,为此推测,内质网功能紊乱所造成的内质网应激是促使NAFLD病程进展的一个重要因素。上述研究结果为进一步阐明NAFLD发生发展的分子机制,发现分期分型诊断标志物和潜在的药物靶点奠定了基础。更多还原

【Abstract】 Nonalcoholic fatty liver disease (NAFLD) has emerged as one of the most common chronic liver diseases in recent years, partly due to the lifestyle changes, including physical inactivity and high daily fat consumption. NAFLD consists of a spectrum of liver disease, ranging from simple steatosis to steatohepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma. NAFLD is strongly associated with obesity, type 2 diabetes and hyperdyslipemia, which are the main features of the recently characterized metabolic syndrome. While a high fat diet is one of the main causes that will induce obesity and its comorbidities. Accumulate evidence suggested that a higher intake of saturated fat can directly affect hepatic fatty infiltration and oxidative damage in different types of liver disease. However, the exact role of saturated fat to the development of NAFLD remains poorly understood. By feeding Sprague-Dawley (SD) rats a high-fat diet (HFD) reproduced many key features of human NAFLD. Current study adopted proteomic approaches to systematically analyze the liver proteomes during the development of HFD-induced NAFLD which may help clarify the pathogenesis of NAFLD.When SD rats were fed with a HFD for 4, 12 and 24 weeks, they successfully reproduced a spectrum of histological changes similar to human NAFLD: that was simple steatosis (SS) at 4 weeks, nonspecific inflammation (NSI) at 12 weeks, and steatohepatitis (NASH) at 24 weeks. Using two-dimensional difference gel electrophoresis (2-D DIGE) combined with MALDI-TOF/TOF analysis, ninety-five proteins exhibiting significant difference (ratio≥1.5 or≤-1.5, P<0.05) during the development of NAFLD were unambiguously identified. This included 53 proteins at SS stage, 42 proteins at NSI stage and 49 proteins at NASH stage. Biological functions of these proteins exhibited phase-specific characteristics during evolution of the disease: at steatosis stage, metabolic enzymes involved in lipid, carbohydrate and amino acid metabolisms were affected highlighted by depressed fatty acidβ-oxidation; by nonspecific inflammation stage, mitochondrial functions were impaired with decreasing proteins involved in mitochondrial respiratory chain and oxidative phosphorylation; up to steatohepatitis stage, cytoskeleton proteins were up-regulated, which may affect hepatocyte structural and functional integrity. Furthermore, a pathway analysis of these proteins identified PPARαand C/EBPαas key regulators that were subsequently verified by Western blot. The results showed that PPARa was decrease at all three stages of NAFLD, while C/EBPαwas increased at the earlier two stages of NAFLD. More importantly, we found that four ER chaperones, PDI, PDIA3, GRP78 and ERp29 were up-regulated especially at the more advanced stages of NAFLD. Up-regulation of ER chaperones is one of the important cellular markers of ER stress. Evidence suggested that ER stress was linked to the development of several diseases including obesity and type 2 diabetes, two major risk factors that strongly associated with NAFLD. Accordingly, we proposed that the disruption of ER function may contribute to the progression of NAFLD from steatosis to NASH. Together, these results help clarify the pathogenesis of NAFLD and identify potential targets for therapeutic interventions.更多还原