【作者】 陶君；

【导师】 蔡卫民；

【作者基本信息】 浙江大学 ， 内科学， 2004， 博士

【摘要】 血吸虫病流行于76个国家和地区。目前估计全球6亿人口受威胁，感染人口1.947亿，每年至少有20000人死于血吸虫病，近年来血吸虫病在我国又有卷土重来的苗头，患病人数居高不下，目前，据专家估计，慢性血吸虫病患者数可达一百万以上，所以，防治血吸虫病的任务依然是十分必要而艰巨的。致日本血吸虫病患者死亡之与严重并发症的主要原因是肝纤维化，以往认为杀虫治病后肝纤维化可完全消失。近年来我们通过动物实验、临床与现场研究，发现血吸虫病肝纤维化患者经彻底杀虫治疗，肝纤维化有好转，但不能完全消除，部分患者肝纤维化程度仍可继续发展，我们推测与肝窦毛细血管化的形成有关。 正常的肝窦壁由一层肝窦内皮细胞(SEC)组成，SEC胞膜不连续，有许多直径100nm左右的小孔，称为窗孔，许多窗孔又组成SEC的肝筛结构。与其它内皮细胞不同的是，SEC的窗孔无隔膜，而且内皮下也无基底膜(BM)存在，肝细胞可与肝窦血液中的营养物质和氧进行自由交换。非血吸虫病肝纤维化研究表明：肝纤维化发生时，肝窦结构发生显著性的改变，即出现SEC持续去窗孔化和BM形成，1963年由Schaffner和Popper首次将此病理现象命名为“肝窦毛细血管化”。肝窦毛细血管化不仅是肝纤维化过程中一个重要病理改变，而且直接影响肝脏病理生理功能，导致肝功能受损，加快肝纤维化的过程。研究肝窦毛细血管化的形成机理以及和肝纤维ZheUniversi2 0 04Ph.D.Dissertation化、肝硬化的发生关系已成为肝病研究领域的一个热点。目前，未见血吸虫病肝窦毛细血管化的研究，而非血吸虫病肝纤维化中(化学性、病毒性等)己有较多的研究，主要有:SEC失窗孔化机制;BM的成分研究;肝窦毛细血管化发生的时间进程;肝窦毛细血管化的后果及肝窦毛细血管化是否可逆等方面。弄清血吸虫病肝纤维化过程中的肝窦毛细血管化不仅有重要的学术价值，而且可能为研究新的血吸虫病有效治疗方法以及判断预后与鉴别诊断提供理论依据。本研究目的首先是证明晚期日本血吸虫病患者肝窦毛细血管化的存在;其次，建立日本血吸虫尾蜘感染BALB/c小鼠肝纤维化模型，观察肝窦毛细血管化动态形成过程及其与肝纤维化的关系等;再次，以杀虫药物毗哇酮和抗肝纤维化药物IFN寺对其干预治疗，探讨它们是否具有逆转血吸虫病肝窦毛细血管化的作用。第一部分晚期日本血吸虫病患者肝窦毛细血管化的研究研究目的:研究晚期日本血吸虫病(晚血)患者是否存在肝窦毛细血管化现象，晚血肝窦毛细血管化与肝纤维化程度、肝损伤的关系。方法:26例晚血患者肝活检和5例正常人肝标本行常规病理染色和天狼猩红染色，进行肝纤维化程度的半定量分析，应用鼠抗人四型胶原(C一IV)单克隆抗体和兔抗人层粘蛋白(LN)多克隆抗体进行免疫组织化学(免疫组化)染色，对肝窦阳性结果进行定量检测，放射免疫法检测血清透明质酸(HA)，自动生化分析仪检测肝功能，对其中5例晚血患者和2例正常人肝新鲜标本进行透射电镜的检查。结果:肝纤维化程度为I级的患者有5例，H级患者有H例，IH级患者有10例，正常人肝细胞浆、肝窦壁和血管内皮细胞有C一W和LN的低水平表达，晚血患者肝组织C一IV和LN的表达除上述部位外，还表达在虫卵肉芽肿周围，肝窦C一IV和LN表达的量明显高于正常人(P均<0.01)，与肝纤维化程度及部分肝功能指标水平相关(尸<0 .05或尸<0.01)，与血清HA水平无关(P>0.05)。透射电镜检查发现正常人SEC有窗孔，SEC下未见BM的形成，晚血患者SEC失窗孔及SEC下可见BM的形成，SEC胞浆内出现weibe!和Palade发现的纺锤状物质(WP小体)，肝窦腔内有脱落的肝细胞微绒毛。结论:1.晚血患者肝组织存在肝窦毛细血管化现象2.晚血患者肝窦毛细血管化与肝纤维化程度及肝功能损伤有关第二部分日本血吸虫感染BLAB/C小鼠肝窦毛细血管化的动态观察研究目的:研究感染日本血吸虫BALB/。小鼠肝窦毛细血管化的动态形成过程，及其与肝纤维化和肝组织损伤的关系，肝窦毛细血管化BM主要成分C一W和LN产生细胞的研究。方法:建立感染日本血吸虫BALB/。小鼠肝纤维化动物模型，肝标本行常规病理染色和天狼猩红染色，进行肝纤维化程度的半定量分析，免疫组化染色检测肝窦C一IV、LN和第姗因子相关抗原(v WF)的表达，测血清ALT和AST指标，另每组取2例感染小鼠和2例正常小鼠新鲜肝组织进行透射电镜的检查。结果:感染日本血吸虫BALB/c小鼠SEC第4周窗孔减少、成，肝纤维化程度随感染时间的延续而逐渐升高，肝窦C一Iv、变小，LN和逐渐增强，血清ALT和AST指标在感染第8周达高峰，在感染第部，第24周又有所上升。第8周BM形vWF的表达也16周回落至底结论:1.血吸虫感染所引起小鼠SEC损伤及其表型改变可能是其诱导肝纤维化重要的始动 机制之一2.感染日本血吸虫第8周小鼠肝窦毛细血管化已基本形成3.肝窦毛细血管化可能具有促进血吸虫病小鼠肝纤维化发展的作用 第三部分 杀虫治疗与抗肝纤维化治疗对日本血吸虫感染BLAB/C小鼠肝窦毛细血管化的影更多还原

【Abstract】 Schistosiomiasis’s epidemic range was among 76 countries and regions. At present, more than 600 million people were threatened, and 194.7 million people were infected. It is estimated that at least 20 thousand people die from schistosomiasis every year. Recently, schistosomiasis seems to reappear around our country and the morbidity still remains high. Now, according to experts, the number of chronic schistosomiasis patients amounts to above 1 million. Thus it is still necessary and tough to supervise and treat schistosomiasis. Liver fibrosis is the main cause leading to death and complication of schistosomiasis japonica. Previously, we thought that liver fibrosis could resolve after chemotherapy with praziquantel. In recent years, according to animal experiments, clinical research and investigation at spot, we found that the degree of liver fibrosis of some patients increased even after treatment.The normal hepatic sinusoid wall consists of sinusoidal endothelia cells (SEC). The membrane of SEC is discrete and there are many core, whose diameters are about 100nm long, called fenestration, in the SEC membrane. Liver sieve structure consists of many fenestrations. Compared with other endothelia cells, SEC has not septum and base membrane (BM) below, thus nutrition and oxygen can transport freely between hepatic cells and liver sinusoid blood. But when liver fibrosis takes place, the fenestrations in SEC are lost and BM formed, which was named for capillarization of liver sinusoid by Schaffner and Popper firstly in 1963. Capillarization of liver sinusoid is not only an vital pathological phenomenon during the development of fibrosis but also plays directly role in the pathophysiological function, which leads to liver function damage and makes liver fibrosisprogress rapidly. It is a focus to study the mechanism of sinsoids capillarization formation and its relationship with liver fibrosis and cirrhosis in liver diseases. At present, there is no study on capillarization of sinusoid in liver fibrosis resulted from schistosomiasis japonica, while studies were widely on liver fibrosis caused by other agents such as chemicals or viruses. The main aspects were the mechanism of loss of fenstration in SEC, elements of base membrane, the course of capillarization of liver sinusoids, the results of capillarization of sinusoids and the question whether capillarization of sinusoids was reversible or not. It has important academic value and may provide theoretical bases for treatment supervision, prognosis evaluation and differential diagnosis to explore mechanism of liver sinusoid capillarization in schistosomiasis japonica. The purposes of our study are firstly to confirm the sinusoid capillarization in the patients with advanced schistosomiasis japonicum, secondly, to establish BALB/c mice liver fibrosis model infected by cercaria of schistosoma japonicum to observe the course of development of sinusoid capillarization and features in pathology. At last, to explore whether sinusoid capillarization is reversible or not after treated with praziquantel and antifibrotic agent (IFN)Part 1Study on liver sinusoid capillarization in the patients with Advanced schistosomiasis JaponicumObjectiveTo study whether liver sinusoid capillarization really existed in the patients with advanced schistosomiasis japonicum and explore its relationship with the degree of hepatic fibrosis and liver function.MethodsLiver biopsy specimens were examined pathomorphologically in 26 patients with advanced schistosomiasis japonicum and 5 healthy to get to know the degree of liver fibrois by semi-quantity. Immunohistochemisty staining was used to detect the positive results by location and quantity with mouse anti human C-IV monoclonal antibody and rabbit anti human LNpolyclonal antibody. Serum HA was measured by RIA and some liver function indexes were detected by automatic biochemistry analyzer, Among them, the ultramicrostructure of the fresh specimens of 5 patients and 2 healthy were observed by transmission electron mic更多还原