【作者】 牛丽文；

【导师】 杨镇；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 第一部分小鼠日本血吸虫病肝纤维化实验模型的构建目的:构建稳定的、类似人类日本血吸虫病肝纤维化自然病程的小鼠日本血吸虫病肝纤维化模型,观察小鼠日本血吸虫病肝纤维化形成的动态过程,为探讨日本血吸虫病肝纤维化发病机制和干预性实验用药时机的选择及疗效观察提供实验依据。方法:血吸虫尾蚴敷贴法感染小鼠,模型组分别于感染日本血吸虫尾蚴后2 wk、4 wk、6 wk、8 wk、12 wk、16 wk、20 wk、24 wk,采用常规HE染色及透射电子显微镜检查对肝组织进行病理组织学检查,常规Van Gieson染色观察感染不同时期肝脏胶原分布部位和含量以及肝纤维化程度的动态变化。结果:HE和VG染色显示小鼠日本血吸虫病肝纤维化模型建立成功,病理证实小鼠日本血吸虫感染成功率100%,完成实验的动物肝纤维化成功率100%。日本血吸虫感染后第6 wk,急性虫卵肉芽肿形成,第8 wk时肉芽肿达到高峰,电镜下,肉芽肿周围的肝细胞内有脂滴形成,发生变性、坏死。之后大部分被吸收而变为具有增殖活性的假结核性虫卵肉芽肿,体积逐渐减小,至感染后第20～24 wk,呈现血吸虫病特有的干线型肝纤维化。日本血吸虫感染第8 wk后,在肝脏虫卵肉肿内及周围可见少量胶原纤维分布;随后,肝脏组织胶原含量和肝纤维化程度随病程发展呈进行性增多或加重。结论:日本血吸虫尾蚴感染的小鼠日本血吸虫病肝纤维化模型成功再现了人类日本血吸虫病肝纤维化发病的自然进程,为进一步探讨日本血吸虫病肝纤维化发病机制和抗肝纤维化治疗奠定了实验基础。第二部分瘦素受体在小鼠日本血吸虫病肝纤维化组织中的动态表达及作用的实验研究目的:观察小鼠感染日本血吸虫后不同时期肝脏瘦素长受体(OB-Rb)mRNA和蛋白动态表达,探讨瘦素及其受体在日本血吸虫病肝纤维化发生发展中的作用。方法:日本血吸虫尾蚴皮肤敷贴法感染小鼠构建日本血吸虫病肝纤维化模型,于感染后不同时间取肝脏标本。HE和VG染色对肝组织进行病理学检查,免疫组织化学SP法动态观察OB-Rb及肝星状细胞(HSC)标记物α-平滑肌动蛋白(α-SMA)表达;逆转录-聚合酶链反应(RT-PCR)检测OB-Rb mRNA动态表达。结果:α-SMA和OB-Rb蛋白阳性表达随病程进展进行性增加,二者共表达于胶原纤维沉积处;模型组OB-Rb mRNA在感染4 wk开始表达,随病程进展逐渐增高,持续至24 wk。相关分析表明,α-SMA蛋白表达与胶原含量(r=0.956, P<0.01)及肝纤维化程度(r=0.804, P<0.01)均呈正相关;OB-Rb蛋白和mRNA表达与胶原含量(r=0.965, P<0.01;r=0.945, P<0.01)及肝纤维化程度(r=0.823, P<0.01; r=0.880, P<0.01)均呈正相关。结论:瘦素、HSC和日本血吸虫病肝纤维化之间有着极为密切的关系,HSC是日本血吸虫病肝纤维化发生发展过程中的关键细胞,而瘦素可能是日本血吸虫病肝纤维化形成过程中一个新的促肝纤维化因子。第三部分瘦素在小鼠日本血吸虫病肝纤维化作用的分子机制的实验研究目的:观察小鼠感染日本血吸虫后不同时期肝脏瘦素和磷脂酰肌醇-3激酶(PI3K)蛋白及α1(I)型前胶原mRNA在日本血吸虫病肝纤维化形成过程中的动态表达,研究PI3K通路在瘦素促进日本血吸虫病肝纤维化过程中的作用,初步阐明瘦素致日本血吸虫病肝纤维化的分子机制。方法:日本血吸虫尾蚴皮肤敷贴法感染小鼠构建日本血吸虫病肝纤维化模型,于感染后不同时间取肝脏标本。HE和VG染色对肝组织进行病理学检查,免疫组织化学SP法动态观察瘦素和PI3K蛋白表达;Western Blot法分析PI3K蛋白的动态表达; RT-PCR检测α1(I)型前胶原mRNA动态表达。结果:瘦素和PI3K蛋白阳性表达随病程进展进行性增加,二者共表达于胶原纤维沉积处;模型组PI3K在感染4 wk开始表达,随病程进展逐渐增高,持续至24 wk。相关分析表明,瘦素蛋白表达与肝脏胶原含量(r=0.758,P<0.01)、α1(I)型前胶原mRNA(r=0.832 ,P<0.01)及肝纤维化程度均呈正相关(r=0.823,P<0.01);PI3K蛋白表达与瘦素蛋白(r=0.882,P<0.01)、肝脏胶原含量(r=0.889,P<0.01)、α1(I)型前胶原mRNA(r=0.708,P<0.01)及肝纤维化程度(r=0.807,P<0.01)均呈正相关;α1(I)型前胶原mRNA与胶原含量(r=0.824,P<0.01)、肝纤维化程度均呈正相关(r=0.612,P<0.01)。结论:瘦素通过增强PI3K磷酸化,而促进α1(I)型前胶原mRNA表达和胶原合成,进而促进日本血吸虫病肝纤维化的形成,PI3K通路在该过程中发挥重要作用。第四部分维生素E对小鼠日本血吸虫病肝纤维化的治疗作用及其机制的实验研究目的:观察维生素E(VitE)对α-SMA、瘦素和PI3K蛋白以及α1(I)型前胶原基因表达的影响,探讨VitE抗小鼠日本血吸虫病肝纤维化作用及其机制。方法:日本血吸虫尾蚴皮肤敷贴法感染小鼠构建日本血吸虫病肝纤维化模型,以肝纤维化达Ⅱ级或Ⅱ级以上作为开始治疗的标志。随机分5组:正常对照组、模型组以及VitE高、中、低剂量组(150 mg/kg、50 mg/kg、5 mg/kg)。于第8 wk末处死动物,HE和VG染色以及透射电子显微镜对肝组织进行病理学检查,应用分光光度法检测肝组织丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性,采用免疫组织化学SP法检测HSC标记物α-SMA和瘦素蛋白表达情况,Western Blot方法分析PI3K蛋白表达情况,RT-PCR方法检测肝脏α1(I)型前胶原基因表达。结果:VitE降低模型组肝组织MDA含量(P<0.01),提高SOD活性(P<0.01);减少α-SMA和瘦素阳性表达细胞数(P<0.01);降低肝脏PI3K蛋白表达(P<0.01)、胶原含量(P<0.01)和α1(I)型前胶原基因表达(P<0.01),均呈剂量效应关系。结论:VitE具有抗小鼠日本血吸虫病肝纤维化作用,其机制与VitE抗脂质过氧化作用、抑制HSC活化和增殖、抑制瘦素表达、阻断PI3K通路、降低α1(I)型前胶原基因表达和胶原合成有关。更多还原

【Abstract】 Part I Construction and identification of liver fibrosis of mice with Schistosoma JaponicaObjective: To construct mice models with Schistosoma Japonica, reliable and resemble to human liver fibrosis,and to observe the progress of liver fibrosis, and to provide for further investigation on pathogenesy and treatment of liver fibrosis of Schistosomiasis Japonica. Methods: Mice were infected with Schistosoma Japonicum cercariae percutaneously, and liver biopsies were done at different time point after infection(2 wk、4 wk、6 wk、8 wk、12 wk、16 wk、20 wk、24 wk). Liver lesions were evaluated using HE staining and transmission electron microscopy. The dynamic changes of liver collagen location and content as well as liver fibrosis degree at different time point after infection were evaluated by Van Gieson staining.Results: All Mice infected with Schistosoma Japonica were confirmed by HE and VG staining, and all animals had been constructed for models of hepatic fibrosis. Granulomae were found in liver at 6 weeks after infection, and achieved the peak of the quantity and size at 8 weeks after infection, and fewer and smaller because of transforming to tubercle-like node from then on. During 20 to 24 weeks after infection, the typical argilla-pipe hepatic fibrosis was observed. A small quantity of liver collagen was detected in and around granulomae. From then on, collagen content and degree of liver fibrosis increased or aggravated progressively with course of diseases.Conclusion: Hepatic fibrosis models of mice with Schistosoma Japonica can show reliably the progress of human hepatic fibrosis of Schistosomiasis Japonica, which lays the foundation for further study on pathogenesy and treatment of liver fibrosis of Schistosomiasis Japonica.PartⅡThe experimental studies on dynamic changes of OB-Rb mRNA and protein expression and their significance in liver fibrosis of mice with Schistosoma JaponicaObjective: To observe the dynamic changes in the long form of leptin receptor(OB-Rb) mRNA and protein expression in mice liver by Schistosoma Japonica infection at different period, and to investigate the roles of leptin and its receptor in liver fibrosis of mice with Schistosoma Japonicum.Methods: Mice infected with Schistosoma Japonica cercariae percutaneously were served as animal models of liver fibrosis, and liver biopsies were done at different time point after infection. Liver lesions were evaluated using HE and VG staining. Immunohistochemistry for OB-Rb andα-smooth muscle actin(α-SMA) as an activated hepatic stellate cell(HSC) marker was performed to observe their dynamic changes by SP technique. OB-Rb mRNA dynamic expression was measured by RT-PCR.Results:Theα-SMA and OB-Rb positive cells increased gradually with course of diseases. Co-localization of OB-Rb andα-SMA in activated HSCs was in the localization of collagen fibers. OB-Rb mRNA expression in model group began at the 4th week after infection and increased progressively and persisted at the 24th week after infection. There was a positive correlation betweenα-SMA protein expression and collagen content(r=0.956, P<0.01) or degree of liver fibrosis(r=0.804, P<0.01). Similarly, there was a positive correlation between OB-Rb protein or mRNA expression and collagen content(r=0.965, P<0.01; r=0.945, P<0.01) or degree of liver fibrosis(r=0.823, P<0.01; r=0.880, P<0.01).Conclusion: The close relationships exist between leptin, HSC and liver fibrosis by Schistosomiasis Japonica. HSC is a key cell and leptin is a novel profibrogenic cytokine in the development of liver fibrosis by Schistosomiasis Japonica.PartⅢThe experimental studies on the molecular mechanisms of leptin in liver fibrosis of mice with Schistosoma JaponicaObjective: To observe the dynamic changes in leptin and phosphatidylinositol 3-kinase(PI3K) protein expression as well asα1(I) procollagen mRNA expression in mice liver by Schistosoma Japonica infection at different period, and to investigate the roles of PI3K pathway in liver fibrosis of mice with Schistosomiasis Japonica by leptin. Methods: Mice infected with Schistosoma Japonica cercariae percutaneously were served as animal models of liver fibrosis, and liver biopsies were done at different time point after infection. Liver lesions were evaluated using HE and VG staining. Immunohistochemistry for leptin and PI3K was performed to observe their dynamic changes by SP technique. PI3K protein dynamic expression was detected by Western Blot, andα1(I) procollagen mRNA dynamic expression was measured by RT-PCR.Results:The leptin and PI3K positive cells increased gradually with course of diseases. Co-localization of leptin and PI3K in activated HSCs was in the localization of collagen fibers. PI3K protein expression began at the 4th week after infection and increased progressively and persisted at the 24th week after infection. There was a positive correlation between leptin protein expression and collagen content(r=0.758, P<0.01) orα1(I) procollagen mRNA(r=0.832, P<0.01) or degree of liver fibrosis(r=0.823, P<0.01). Similarly, there was a positive correlation between PI3K protein and leptin(r=0.882, P<0.01) or collagen content(r=0.889, P<0.01) orα1(I) procollagen mRNA(r=0.708, P<0.01) or degree of liver fibrosis(r=0.807, P<0.01). There was a positive correlation betweenα1(I) procollagen mRNA and collagen content(r=0.824, P<0.01) or degree of liver fibrosis(r=0.612, P<0.01).Conclusion: Leptin promotedα1(I) procollagen mRNA expression and protein production by stimulating PI3K phosphorylation in the progress of liver fibrogenesis of mice with Schistosomiasis Japonica, and PI3K pathway has an important effect in the process.PartⅣThe experimental studies on therapeutic effect of vitamin E and its mechanisms on liver fibrosis of mice with Schistosoma JaponicaObjective: To observe the effect of vitamin E(VitE) onα-SMA, leptin and PI3K protein expression as well asα1(I) procollagen mRNA expression, and to investigate the therapeutic roles of VitE and its mechanisms in liver fibrosis of mice with Schistosoma Japonica. Methods: Mice were infected with Schistosoma Japonicum cercariae percutaneously. That liver fibrosis reached gradeⅡor above was considered to be a marker of therapeutic start.Mice were randomly divided into five groups: normal control group, model group, and intervention groups which were treated with three different doses of VitE, including 150 mg/kg, 50 mg/kg, and 5 mg/kg. The mice were killed at the end of the eighth week. Liver lesions were evaluated using HE and VG staining as well as transmission electron microscopy. The malondialdehyde(MDA) content and superoxide dismutase(SOD) activity in liver tissue were determined by spectrophotometric method. Immunohistochemistry forα-SMA as an HSC marker and leptin was performed by SP technique. PI3K protein expression was detected by Western Blot, and theα1(I) procollagen mRNA expression was measured by RT-PCR.Results:VitE reduced MDA content(P<0.01) and increased SOD activity(P<0.01) in the liver in model group in a dose-dependent manner. Besides, VitE decreased the number ofα-SMA(P<0.01) and leptin(P<0.01) positive cells in a dose-dependent manner. Further, VitE diminished increased PI3K protein expression(P<0.01), collagen content(P<0.01) and inhibited increasedα1(I) procollagen mRNA expression(P<0.01) in the liver in model group in a dose-dependent manner.Conclusion: VitE has evident therapeutic effects on liver fibrosis produced in mice by Schistosoma Japonicum infection, and the mechanisms are associated with VitE opposing lipid peroxidation, inhibiting HSC activation and proliferation, inhibiting leptin protein expression, blocking PI3K pathway, and reducingα1(I) procollagen mRNA expression and collagen production.更多还原