【作者】 李岽健；

【导师】 杨镇；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 目的:1、构建稳定、可靠、符合血吸虫感染自然演变过程的血吸虫病门脉高压小鼠动物模型,研究该模型肝脏病变和门静脉高压的进展情况。2、研究血吸虫病门脉高压小鼠肠黏膜屏障功能的改变和内毒素血症的关系,探讨内毒素通过肠黏膜屏障的机制。3、研究LPS信号转导通路的跨膜受体TLR-4/MD-2在血吸虫病门脉高压小鼠肠系膜静脉中的表达,以探讨肠源性内毒素血症在门脉高压性肠系膜静脉血管病变中的作用。4、研究IRAK-4在血吸虫病门脉高压小鼠肠系膜静脉中的表达情况,探讨LPS导致门脉高压性肠系膜静脉血管病变的机制。方法:1、用腹部敷贴法制作血吸虫病小鼠模型,分别在感染尾蚴后6周、12周、20周,以HE染色、Masson三色染色、透射电镜观察小鼠肝脏及结肠组织病变情况,测量门静脉直径和门静脉压力以了解该模型血液动力学变化情况。2、用FITC-D法检测血吸虫病门脉高压小鼠肠黏膜通透性的改变,MT-1鲎试剂基质显色法测定门静脉血内毒素含量,取肝脏组织细菌培养以了解细菌移位情况;免疫组织化学和Western blot法检测结肠组织occludin蛋白表达,TUNEL法检测结肠黏膜细胞原位凋亡。3、RT-PCR方法检测血吸虫病门脉高压小鼠肠系膜静脉中TLR-4/MD-2及TNF-α的表达,将门静脉血内毒素含量与TLR-4/MD-2表达量行相关性分析。4、Western blot和RT-PCR检测IRAK-4在血吸虫病门脉高压小鼠肠系膜静脉中的表达,行IRAK-4和TNF-α表达的相关性分析。结果:1、腹部敷贴法制作血吸虫病小鼠模型,感染成功率100%,死亡率36%;动物感染后12周出现肝纤维化,20周出现肝硬化表现;门静脉压力在感染后12周就明显升高,并持续保持较高水平。2、与正常组对比,随着病程的进展,各时间组小鼠门静脉血FITC-D含量显著升高(p<0.01),门静脉血中内毒素含量及肝组织细菌培养均同步上升,门静脉血中内毒素含量与FITC-D含量呈显著正相关(r=0.543,p<0.01);紧密连接蛋白occludin表达显著减少;肠黏膜细胞原位凋亡明显增多。(p值均<0.01)3、血吸虫病小鼠肠系膜静脉中TLR-4/MD-2及TNF-α的mRNA表达均较正常组显著升高(p<0.01);20周组TLR-4/MD-2表达与12组比较显著升高(p<0.01),TLR-4/MD-2表达量与门静脉血内毒素含量呈正相关(r1=0.548, p<0.01;r2=0.534, p<0.01);TNF-α的表达12周组与20周组无显著性差异(p>0.05)。4、各时间组血吸虫病小鼠肠系膜静脉IRAK-4 mRNA和蛋白的表达较正常组显著升高(p<0.01),12周组与20周组间比较无显著性差异(p>0.05),IRAK-4表达量与TNF-α呈正相关(r=5.56, p<0.01)。结论:我们所构建的血吸虫病小鼠模型符合人体血吸虫病的发病规律、门静脉系统血流动力学改变明显,是研究血吸虫病及门脉高压症的理想模型;血吸虫病门脉高压小鼠随着门脉压力的增高,存在着明显的肠黏膜屏障功能障碍和肠源性内毒素血症,肠道通透性升高是肠源性内毒素血症产生的重要原因之一。细菌、内毒素等大分子物质可能通过细胞旁途径和跨细胞途径进入肠道固有层和门静脉血;入血后的内毒素通过其特异性的TLR-4信号转导途径启动对肠系膜静脉的损害,引起和加重门脉高压性血管病变;但其下游TNF-α等炎性因子表达与TLR-4表达量无明显相关,而IRAK-4可能才是内毒素诱导的炎性因子表达的关键性调控因子,其在内毒素导致的门脉高压肠系膜血管病变中起着重要作用,是治疗门脉高压性血管病变的一个有效靶点。更多还原

【Abstract】 OBJECTIVE1. Design a steady、credible rat model of schistosomal portal hypertension. The model accord with the nature course of schistosomiasis. To observe the change of liver criihosis and portal hypertension.2. To study the relation of gut barrier and endotoxemia. To explore the track of endotoxin through gut barrier.3. To study the changes of expression of Toll-like receptor 4 and myeloid differentiation protein-2 of mesenteric vein in rats with Schistosomal portal hypertension. To explore the role of intestinal endotoxemia in mesenteric vein vasculopahty.4. To study the changes of expression of interleukin 1 receptor-associated kinase 4 of mesenteric vein in mice with Schistosomal portal hypertension.METHODS1. Infected mice percutaneously with cercariae of schistosomiasis japonica. Subdividing mice into 6 week、12 week and 20 week groups. HE stain、Masson trichrome stain and transmission electron microscope examination was used to observe pathologic pattern of liver and colon. The diameter of portal vein and portal venous pressure was used to observe the change of flow dynamics.2. To observe the change of gut barrier function by FITC-D. Endotoxin was measured by MT-1 reagent box. Liver tissue was collected for germiculture. Immunohistochemistry and quantitative analysis of Western blot were applied to detect the protein expression of occluding. TUNEL methods was applied to detect the apoptosis of colon mucous membrane cell.3. RT-PCR were applied to detect the mRNA expression of TLR-4/MD-2 and TNF-αin the mesenteric vein. Analyzed the pertinency between expression of TLR-4 and endotoxemia.4. RT-PCR and Western blot were applied to detect the protein and mRNA expression of IRAK-4 in the mesenteric vein. Analyzed the pertinency between expression of IRAK-4 and TNF-α.RESULTS1. The infected rate of model mice is 100%, mortality rate is 36%. At the 12th week after infection, the liver fibrosis was occurred; at the 20th week after infection, the liver cirrhosis was occurred. Portal venous pressure was gradually raised at 12th week group, and keeping a high level at 20th week group.2. The concentration of FITC-D and in 12th week and 20th week group is significantly stronger than normal group (p<0.01), this trend was positively correlated to increased the concentration of endotoxin(r=0.543,p<0.01). The ratio of bacteria translocation in 12th week and 20th week group is also significantly higher than normal group (p<0.01). The expression of occludin in 12th week and 20th week group is significantly lower than normal group; The apoptosis index of 12th week and 20th week group is significantly higher than normal group.3. The expression of TLR-4、MD-2 and TNF-αmRNA in 12th week and 20th week group is significantly stronger than normal group (p<0.01). The expression of TLR-4/MD-2 mRNA was positively correlated to increased of the concentration of endotoxin(r1=0.548, p<0.01;r2=0.534, p<0.01). The expression of TNF-αmRNA in 12th week has no significantly difference(p>0.05).4.The expression of IRAK-4 protein and mRNA in 12th week and 20th week group is significantly stronger than normal group (p<0.01), comparing 12th week and 20th week group, there has no significantly difference(p>0.05). The expression of IRAK-4 mRNA was positively correlated to increased of the expression of TNF-αmRNA(r=5.56, p<0.01). CONCLUSIONThe mice model establish by infecting percutaneously with cercariae of schistosomiasis japonica has a nature course of schistosomiasis. It has a significantly change of portal flow dynamics. It is a appropriate model to study schistosomal cirrhosis and portal hypertension. The development of portal hypertension is accompanied by the damaged of gut barrier and portal endotoxemia. The raise of intestinal permeability is the main reason of intestinal endotoxemia. LPS can upregulate the expression of its signaling receptor :TLR-4. Through the TLR-4 signaling pathways, LPS can induce IRAK-4 is the key regulator in the TLR-4 signaling pathway, it play an important roll in portal hypertension vasculopahty.更多还原