原发性胆汁性肝硬化发病机制研究及药物治疗探索

【作者】 刘斌；

【导师】 张奉春；

【作者基本信息】 中国协和医科大学 ， 风湿病学， 2008， 博士

【摘要】 第一部分原发性胆汁性肝硬化小鼠模型的建立和鉴定原发性胆汁性肝硬化(Primary biliary cirrhosis,PBC)是以胆管上皮损伤为特征的器官特异性自身免疫性肝病,最终导致胆汁淤积、肝脏纤维化甚至肝硬化。目的:本研究拟应用干扰素-1诱导剂,聚肌胞(polyinosinic polycytidylic acid,polyI:C),诱导PBC样自身免疫过程,从而建立PBC动物模型。方法:6到8周雌性C57BL/6小鼠腹腔注射5 mg/kg的poly I:C,每周两次;于第8周、第16周、第24周收集小鼠肝脏、血清标本,病理检测及免疫组化分析肝脏淋巴细胞浸润程度。间接免疫荧光(immunofluorescence,IIF)和酶联免疫吸附试验(enzyme-linkedimmunosorbent assay,ELISA)检测血清抗线粒体抗体(antimitochondrial antibody,AMA)。结果:poly I:C腹腔注射后,第8周C57BL/6小鼠肝脏开始出现门脉汇管区炎症细胞浸润,随着造模时间的延长到第16周小鼠肝脏出现小胆管增生,第24周出现胆汁淤积。第24周时所有poly I:C腹腔注射小鼠血清AMA均阳性,并出现碱性磷酸酶和总胆红素升高。结论:通过poly I:C腹腔注射可使遗传易感雌性C57BL/6小鼠出现PBC样改变,为PBC发病机制的研究及其新药物疗效的评价提供帮助。第二部分TGF-β1信号传导通路异常与原发性胆汁性肝硬化的发生发展原发性胆汁性肝硬化(Primary biliary cirrhosis,PBC)以肝内小胆管进行性破坏伴门脉炎症性改变、抗线粒体抗体(anti-mitochondrial antibodies,AMAs)阳性为特征的自身免疫性肝病。最新的研究显示转化生长因子β1(transforming growthfactorβ1,TGF-β1)信号传导通路在PBC的发病中起重要作用。目的:研究TGF-β1信号传导通路在PBC发病中所起的作用。方法:6到8周雌性C57BL/6小鼠(n=6)腹腔注射聚肌胞(poly I:C)复制PBC模型,免疫组化、免疫印迹和实时定量PCR分析模型组小鼠和对照组小鼠肝脏TGF-β1、TGF-β受体Ⅰ(TβRⅠ)、TGF-β受体Ⅱ(TβRⅡ)、p-Smad2/3、α平滑肌肌动蛋白(α-SMA)和α1(Ⅰ)型胶原的表达。流式细胞仪分析小鼠肝脏、脾脏淋巴细胞亚群。结果:模型组小鼠具有包括碱性磷酸酶(ALP)升高、AMA阳性、肝内小胆管进行性破坏伴门脉炎症性改变在内的类似人类PBC的特征。免疫组化显示,PBC模型组小鼠肝脏TGF-β1、TβRⅠ、TβRⅡ、p-Smad2/3、α-SMA和α1(Ⅰ)型胶原蛋白表达明显增高;免疫印迹和实时定量PCR显示,PBC模型组小鼠肝脏TGF-β1、TβRⅠ、TβRⅡ、p-Smad2/3、α-SMA和α1(Ⅰ)型胶原蛋白和mRNA水平高于正常对照小鼠(P＜0.05)。PBC模型组小鼠肝脏、脾脏CD4~+CD25~+FOXP3~+和CD8~+T细胞总数和比例均高于正常对照组小鼠(P＜0.01),且与TGF-β1升高有关。结论:TGF-β1在PBC的发生发展中可能起双重作用:TGF-β1在抑制炎症反应的同时导致肝脏纤维化。TGF-β1信号传导通路的异常对PBC的发生发展中起着重要作用。第三部分姜黄预防和改善原发性胆汁性肝硬化小鼠模型病变的发展姜黄有抗炎、抗氧化和抗纤维化活性,最新的研究显示姜黄有益于慢性肝病的治疗。目的:通过本研究探讨姜黄是否可以预防和减轻原发性胆汁性肝硬化(PBC)小鼠门脉区炎症和胆管损害。研究其治疗机制是否为通过增加过氧化物酶体增生物激活受体(peroxisome proliferator-activated receptorγ,PPARγ)活性抑制转化生长因子-β1(transforming growth factor-β1,TGF-β1)所致。方法:6到8周雌性C57BL/6小鼠(n=6)腹腔注射聚肌胞(poly I:C)复制PBC模型,同时分别给以姜黄(200、400和800mg/kg/d),或容积灌胃治疗。比较对照组和各治疗组之间肝脏生化、抗线粒体抗体(anti-mitochondrial antibodies,AMAs)和肝脏病理组织改变。通过免疫组化、免疫印迹和实时定量PCR分析对照组和治疗组之间小鼠肝脏TGF-βl、TGF-β受体Ⅰ(TβRⅠ)、TGF-β受体Ⅱ(TβRⅡ)、p-Smad2/3、PPARγ、α平滑肌肌动蛋白(α-SMA)和α1(Ⅰ)型胶原变化。结果:姜黄400和800mg治疗组小鼠肝脏生化、AMAs阳性率和肝脏病理组织改变均较PBC模型组明显改善,差距有显著统计学意义(P＜0.05)。免疫组化显示,姜黄400和800mg治疗组小鼠肝脏TGF-β1、TβRⅠ、TβRⅡ、p-Smad2/3、PPARγ、α-SMA和α1(Ⅰ)型胶原表达较PBC模型组小鼠降低,而PPARγ表达增加。免疫印迹和实时定量PCR显示,姜黄400和800mg治疗组小鼠肝脏TGF-β1、TβRⅠ、TβRⅡ、p-Smad2/3、α-SMA和α2(Ⅰ)型胶原蛋白和mRNA水平低于PBC模型组小鼠(P＜0.05),而PPARγ表达高于PBC模型组,差距有显著统计学意义(P＜0.05)。结论:姜黄可能通过剂量依赖性活化PPARγ抑制TGF-β1信号通路,预防和改善PBC模型组小鼠肝脏病变发展,可能成为治疗PBC的新药物。更多还原

【Abstract】 Section one Establish and identify the animal model of primary biliary cirrhosisBackground:Primary biliary cirrhosis(PBC) is one of the organ specific autoimmune diseases characterized by destruction of the biliary epithelial cells, cholestasis,liver cirrhosis,and liver failure.With the postulation that induction of the autoimmune process might induce PBC-like cholangitis,here we used polyinosinic polycytidylic acid(poly I:C),an inducer of type-1 interferon(IFN),to generate an autoimmune cholangitis animal model.Methods:Female C57BL/6 mice were injected with 5 mg/kg of poly I:C twice a week for 24 consecutive weeks.Liver specimens were collected to evaluate the degree of cell infiltration.Autoantibodies, including antimitochondrial antibodies(AMAs),were assayed by immunofluorescence (IIF) and enzyme-linked immunosorbent assay(ELISA).Results:Mononuclear cells were detected at the portal areas 8 weeks after the start of poly I:C injection,which progressed up to 24 weeks.Autoantibodies,including AMAs,were detected in the sera from all poly I:C injected mice.Conclusions:A PBC-like cholangitis in a genetically susceptible mouse strain because of poly I:C administration.This model would be helpful to study PBC immunopathogenesis and to evaluate the effectiveness of newly developed therapeutic regimens for PBC. Section two Aberrant TGF-β1 signaling contributes to the development of primary biliary cirrhosis in murine modelBackground:Primary biliary cirrhosis(PBC) is an autoimmune liver disease, characterized by lymphocytic infiltration in portal tracts,selective destruction of biliary epithelial cells,and anti-mitochondrial antibodies(AMAs).Recent studies suggest that TGF-β1 signaling pathway might play an important role in the pathogenesis of PBC.Aims:To investigate whether TGF-β1 signaling pathway is involved in the pathogenesis of PBC.Methods:A murine model of PBC was developed by injection of polyinosinic polycytidylic acids(poly I:C) in C57BL/6 mice,and the liver expressions of TGF-β1,TGF-βreceptorⅠ(TβRⅠ),TGF-βreceptorⅡ(TβRⅡ),p-Smad2/3,α-smooth muscle actin(α-SMA) andα1(Ⅰ) collagen in mice model and control mice were evaluated by immunohistochemistry,immunoblotting and semi-quantitative real-time PCR.Lymphocyte subsets in liver and spleen were analyzed using flow cytometry.Results:The mice model had several key phenotypic features of human PBC,including elevated levels of alkaline phosphatase(ALP), AMAs,portal bile ducts inflammation,and progressive collagen deposition.Protein and mRNA levels of TGF-β1,TβRI,TβRⅡ,p-Smad2/3,α-SMA andα1(Ⅰ) collagen were higher in liver from mice model compared with that in control mice(P＜0.05),as well as the total number and percentage of CD4~+CD25~+FOXP3~+ and CD8~+ lymphocytes(P＜0.01).Conclusion:TGF-β1 might play a dual role in the development of PBC:it suppresses inflammatory response but operates to enhance fibrogenesis on the other hand.The aberrant activity of TGF-β1 signaling contributes to the development of PBC. Section three Curcumin Prevents and Ameliorates the development of primary biliary cirrhosis in murine modelBackground & Aims:Curcumin is well documented to have a variety of beneficial effects,including antioxidative,anti-inflammatory and anti-fibrosis activities.Recent evidence suggests that it may be of therapeutic interest in chronic liver disease.The aim of this study was to determine whether treatment with curcumin prevented and ameliorated portal inflammation in a murine model of primary biliary cirrhosis(PBC). We also tested whether inhibition of transforming growth factor-β1(TGF-β1) via peroxisome proliferator-activated receptorγ(PPARγ) by curcumin was involved in these mechanisms.Methods:A murine model of PBC developed by injection of polyinosinic polycytidylic acids(poly I:C) in C57BL/6 mice were treated with curcumin(200,400 or 800 mg/kg/day),or vehicle by gavage.Anti-mitochondrial antibodies(AMAs),biochemical variables together with liver histology were evaluated.TGF-β1,TβRⅠ,TβRⅡ,p-Smad2/3,PPARγ,α-SMA andα1(Ⅰ) collagen were evaluated using immunohistochemistry,immunoblotting and semi-quantitative real-time PCR in liver.Results:Treatment of mice with curcumin prevented and improved both biochemical variables and histopathologic signs of murine model of PBC.Consistent with these findings,T-cell infiltration and TGF-β1 signaling pathway activity in liver were suppressed in the curcumin-treated group.Activation of PPARγin liver was also observed.Conclusions:This study has shown for the first time that treatment with curcumin can prevent and improve murine experimental PBC.This finding suggests that curcumin could be a potential therapeutic agent for the treatment of patients with primary biliary cirrhosis.更多还原