【作者】 陈鹏；

【导师】 王文林； 陈元晓； 雷霖； 田明；

【作者基本信息】 昆明医学院 ， 病原生物学， 2009， 硕士

【摘要】 [目的]观察斯氏狸殖吸虫对大鼠肝脏损害的不同时期,肝脏纤维化与细胞因子IL-4、INF-γ在肝脏表达的动态变化关系,探讨IL-4、IFN-γ在斯氏狸殖吸虫感染大鼠肝脏纤维化形成及调节中的作用和意义,从而了解Th1/Th2在斯氏狸殖吸虫感染大鼠肝纤维化形成及调节作用,进一步探索斯氏狸殖吸虫感染大鼠肝纤维化中的细胞免疫应答机制。[方法]将36只大鼠随机分为两组,即对照组6只和感染组30只。将斯氏狸殖吸虫囊蚴经腹腔注射感染大鼠以建立动物模型。感染组则按感染时间的长短分为A组(感染1周),B组(感染2周),C组(感染4周),D组(感染8周),E组(感染12周),F组(感染16周)。分别在感染后的不同时段剖杀大鼠,取其肝脏,采用石蜡切片,HE染色、VG染色,光镜观察肝脏坏死程度及胶原分布情况;免疫组织化学法测定IL-4、IFN-γ在斯氏狸殖吸虫病大鼠肝脏肝纤维化形成过程中的表达动态变化。[结果]HE染色和VG染色结果:对照组正常肝组织的肝细胞排列整齐,肝小叶结构完整,仅见少许纤细纤维分布于血管壁;汇管区无纤维结缔组织增生,无炎性细胞浸润,免疫组化方法观察正常组肝脏细胞因子IL-4、IFN-γ在肝脏基本不表达。感染组感染后1周末肝小叶结构完整,肝细胞水肿;感染后2周末大鼠肝小叶结构完整,出现广泛的肝细胞脂肪变性及散在的气球样变性,中央静脉及汇管区有大量炎症细胞浸润,并可见少数点状坏死,VG染色无纤维结缔组织增生;感染4周末大鼠肝小叶结构仍较完整,但肝细胞气球样变性、坏死及炎症细胞浸润较2周末明显,出现片状坏死,VG染色有少许纤维结缔组织增生;感染后8周末肝脏突出的病变为急性化脓性炎症,在肝组织内形成小脓肿;感染后12周末肝组织内出现片状纤维组织增生,小血管及小胆管增生,部分肝小叶结构破坏、重建,部分门管区纤维增生,增生纤维形成条索,向肝小叶内伸展,增生纤维内可见少数淋巴细胞、中性粒细胞浸润;感染后16周末出现肝细胞大片坏死,汇管区纤维组织增生,增生纤维形成条索,小叶结构紊乱,出现桥接样坏死。感染组各组随着斯氏狸殖吸虫感染时间的长短、纤维化程度的加深、细胞因子IL-4、IFN-γ在肝脏表达有所变化。阳性表达多定位汇管区、血管壁、纤维间隔条索间及肝小叶坏死区的异物巨细胞、淋巴细胞和巨噬细胞的胞浆;肝细胞内未见阳性信号。Th1细胞因子IFN-γ在感染后1到8周末开始上升,在8周末达高峰,随后下降。而Th2细胞因子IL-4在感染后12周末时迅速上升且随感染时间延长而明显升高。[结论]结合肝脏损害HE染色、VG染色的光镜片得出结论:大鼠感染斯氏狸殖吸虫囊蚴后肝脏的损害程度在感染后随着时间的延长纤维化在不断加深,IL-4、IFN-γ随着肝脏纤维化程度的加深表达有所变化。Th1细胞分泌的细胞因子IFN-γ与斯氏狸殖吸虫肝纤维化诱导和形成密切相关。而Th2细胞分泌的细胞因子IL-4可抑制Th1细胞分泌的细胞因子,Th2细胞分泌的细胞因子IL-4在斯氏狸殖吸虫肝纤维化的自发性免疫调节中起重要作用。更多还原

【Abstract】 [Objective]To observe the dynamically changing relationship between hepatic fibrosis and expression of cytokine interleukin-4,interferon-γ,in rats infected with Pagumogonimus skrjabini liver damage of different periods,To explore the function and significance of interleukin-4,interferon-γ,in liver fibrosis formation and regulation of rats infected with Pagumogonimus skrjabini,and thus know the role and significance of the Th1/Th2 in rats infected with Pagumogonimus skrjabini liver fibrosis formation and regulation.To further study the mechanisms of celluar immune response in liver fibrosis of rats infected with Pagumogonimus skrjabini.[Methods]36 rats were randomly divided into two groups:the control group and infection group.There were 6 rats in the control group,30 rats in the infection group.The animal model were set up by the way of peritoneal injection of Pagumogonimus skrjabini metacercaria to infect rats.Infection group were divided into 6 groups according to infection time:A group(infection for 1 week),B group (infection for 2 weeks),C group(infection for 4 week),D group(infection for 8 week),E group(infection for 12 week),F group(infection for 16 week).All rats were killed in corresponding tine points after being infected.then,there liver tissues were fixed,at last,they were made into paraffin sections,HE staining,VG staining,and observed the extent of liver necrosis and collagen distribution by light microscopy meanwhile,measured the dynamic chang of expression of interleukin-4,interferon-γ, in the formed process of liver fibrosis in rats infected with Pagumogonimus skrjabini by the use of immunohistochemical. [Results]In control group,the results of HE staining and VG staining showed liver cells were arranged regularly and the structure of hepatic lobules were integrated in liver tissue.Only there were trifle fibres in the vessel wall,no fiber connective tissue hyperplasia and inflammatory cell infiltration around the portal area.The results of immunohistochemical showed cytokines interleukin-4,interferon-γ,did not almost express in the liver.In infection group,there were hepatocellular edema in the integrated of the structure of hepatic lobules in A group,in B group,there were extensive fatty degeneration and a spot of balloon-like degeneration of liver cells in the structure of hepatic lobules,there were a large number of inflammatory cells infiltration and a small number of pumctiform necrosis in the centralvenous and protal area,no fiber fibrous connective tissue hyperplasia by VG staining.in C group,there were more significant balloon-like degeneration,necrosis and inflammatory cell infiltration in the integrated structure of hepatic lobules than B group,and appeared patchy necrosis,a litter of fiber fibrous connective tissue hyperplasia by VG staining.In D group,the obvious chang of pathology was acute purulent inflammation in liver.A mall obscess was formed in the liver tissue.In E group,the flaky fibrous tissue hyperplasia,the some blood essels and small bile duct hyperplasia appear in liver tissue,the structure of some hepatic lobules were damaged,reconstructed,fiber hyperplasia in part of doortude area.hyperplasia fiber were formed cord.To stretch into hepatic lobules,there were minority lymphocytes,neutrophil infiltration in hyperplasia fiber.In F group,a large of liver cells appeared necrosis,hyperplasia fiber were formed cord,the structure of hepatic lobules a were disorder,then appeared further bridging necrosis.In every group of infection groups,with the infection time,the deepening of the extent of fibrosis, interleukin-4,INF-γexpression in the liver changs.The most positive expressions were positioned in portal area,the vessel wall,fiber spacing between the cord and foreign giant cells,lymphocytes,cytoplasm of macrophages in the necrtic area of hepatic lobules,there were no positive signal in liver cells,interferon-γby Th1 cells secreted began to rise at 1～8weeks after infection,and reached the peak at 8 weeks,then decline.cytokines interleukin-4 by Th2 cells secreted increased rapidly at 12 weeks after infection and with time,the extension of infection increased significantly.[Conclusion]The extent of rats liver damage after infecting with Pagumogonimus skrjabini at the time of fibrosis had been deepened,with the deepening of the degree of liver fibrosis,the expression of interleukin-4,interferon-γwere changed,cytokines interferon-γ,with Thl cells secreting was closely related to induction and the formation of liver fibrosis in rats infected with Pagumogonimus skrjabini,and cytokines interleukin-4 by Th1 cells secreted can repress that Th1 cells secret cytokines,interleukin-4 by Th2 cells secreted plays an important role in spontaneous immune regulation of liver fibrosis in rats infected with pagumogonimus skrjabini.更多还原