【作者】 李春辉；

【导师】 尹正日； 朴东明；

【作者基本信息】 延边大学 ， 病理学与病理生理学， 2003， 硕士

【摘要】 目前认为，反复的肝脏损伤和修复可导致肝星状细胞的增生、激活、转化成成纤维细胞，最终引起细胞外基质(ertracellular matrix,ECM)的过度增多和沉积，从而导致肝纤维化(liver fibrosis,LF)、肝硬化(Cirrhosis)。肝纤维化是肝硬化的早期阶段，是一种可复性的肝脏病变。但目前对其发生机理的认识仍不很清楚。本实验通过低剂量二甲基亚硝胺（Dimethylnitrosamine，DMN）诱发大鼠肝纤维化的模型，用形态学和免疫组织化学等手段对肝纤维化发生机理进行探讨，诣在为临床诊断和治疗肝纤维化提供理论依据。方法：取雄性Wistar大鼠，随机分成五组，即一个对照组和四个中毒组，中毒组腹腔注射DMN(10ul/kg body )一周连续注药三天，持续四周，制作大鼠的肝纤维化模型；对照组在同一时间段内注射同样剂量的生理盐水。大鼠分别在7天、14天、21天、28天称其体重后心脏取血，处死后取其肝脏称其肝重，并计算肝体重比。在不同时间段内用光镜、透射电镜、组织化学、免疫组织化学、生物化学等手段，动态观察在肝纤维化形成过程中肝星状细胞（hepatic stellate cell,HSC）、肝内皮细胞(hepatic sinusoid Endothelial cell,SEC)、肝枯否氏细胞（hepatic Kupffer cell,KC）、肝隐窝细胞（hepatic Pit cell）、肝细胞（hepatocyte,HC）的形态学变化，以及用ELISA(Enzyme-Linked immunosorbent Assay,ELISA)法测定在肝纤维化形成过程中血清透明质酸(hyaluronic,HA)、层粘连蛋白(laninin,LN)、IV型胶原(type IV collagen)的动态变化。统计学资料，用spss10.0统计软件进行统计学处理。 结果：鼠肝的重量在注射DMN后第7天有明显的增加，随后鼠肝的重量逐渐减轻，到第28天达到最小；最大肝体比出现在第14天。<WP=4>光镜及透射电镜观察：DMN用药组第7天在肝小叶中央区出现出血性坏死，以及由于大小单核细胞的侵入导致肝窦的连续性中断和Disse间隙的消失，在电镜下可以见到肝实质的进一步损坏，肝窦壁出现大的窗口(fenestrae)；在肝窦内皮细胞(sinusoidal endothelial cell,SEC)和增大的窦周隙内可观察到红细胞及隐窝细胞(pit cell)；同时，在这一时期肝枯否氏细胞（KC）大量出现，并且呈现分泌致炎因子能力增强的分泌相。随着时间的增加，与纤维间隔形成有关的具有转化特征的肝星状细胞（HSCs）、肌纤维母细胞(myofibroblasts,MFb)出现，一些Pit细胞通过开放的内皮细胞窗口进入Disse间隙，与肝细胞紧密接触，在所有的大鼠肝实质内出现微小的纤维结节，以及红细胞从发生改变的肝窦壁进入Disse间隙。第28天，在所有的大鼠肝实质内出现包含大量的胶原束的纤维间隔，以及肌纤维母细胞、成纤维细胞，并出现肝窦毛细血管化（sinusoidal capilarization）。 免疫组织化学染色：用a-平滑肌肌动蛋白（a-Smooth muscle actin a-SMA），标记活化的肝星状细胞（HSCs）。在正常组，标记物只在汇管区、小叶中央静脉及分散在组织间的成纤维样细胞（fibroblast-like cell）中出现。在DMN组，第7天在肝实质损伤区域内出现被a-SMA标记的大量伸长的成纤维样细胞；在第28天这种被a-SMA标记的细胞数量达到最大值。用五级分级法统计肝组织的纤维化程度，可见正常组与DMN模型组比较有显著性差异(P＜0.05)，DMN各组组间比较有著性差异(P＜0.05)。组织化学染色：用天狼星红（sirius red）染色染胶原纤维。在正常组，胶原纤维只在汇管区和小叶中央静脉内出现。在DMN组，第7天在坏死的区域内出现少量纤细的胶原纤维，随时间的增加，在出现大量出血性坏死及淋巴细胞浸润的区域同时出现细长的纤维束，纤维束与小叶中央静脉相连并出现扩散的纤维，及较粗的纤维间隔。第28天出现粗大的纤维间隔。经图象分析得出胶原纤维面密度百分比正常组与DMN模型组比较有显著性差异（P＜0.05），DMN各组间有显著性差异（P＜0.05）。血清学检测：在不同时间段内DMN模型组大鼠血清的透明质酸（HA）、<WP=5>层粘连蛋白（LN）和IV型胶原（type IV collagen）的含量随时间的增加而增加，对照组与DMN各组比较有显著性差异﹙P＜0.05），并且DMN各组间比较有显著性差异﹙P＜0.05）。经相关分析得出其含量与胶原纤维面密度百分比呈正相关。本论文提示：肝细胞外基质（extracellular matrix，ECM）生成细胞，特别是肝星状细胞，在肝枯否氏细胞和肝细胞分泌的致炎因子的作用下增生、激活、转化成成纤维细胞，造成ECM的过度增多和沉积是导致肝纤维化发生的主要机理之一；肝窦内皮细胞在LF形成过程中形成肝窦毛细血管化，加重肝损伤，促进LF的发展；陷窝细胞可能与其他细胞相互协同来促进LF形成；在LF形成过程中血清中的透明质酸、层粘连蛋白、IV型胶原的含量随LF的发展而升高，并与LF的发展呈正相关更多还原

【Abstract】 Present view , repeated chronic damage to the liver can result proliferation and differentiation of hepatic stellate cells finally cause excess proliferation and doposit of ertracellular matrix (ECM) ,cause fibrosis and cirrhosis. Fibrosis is the stage of cirrhosis and is a recoverable pathologic change..Despite numerous studies on the development of hepatic fibrosis,the mechanism remains unclear this experiment wished to clarily the mechanisms through morphological and biochemical study of the early stages of hepatic fibrosis induced by low does of Dimethylnitrosamine in the rat .Method : male rats of the Wistar strain ,divude into five groups randomly ie: control group and four treatment groups. treatment groups were used for the induction of liver injury.DMN (10ul/kg body weight ,i.p.) was given 3 consecutive days a week for 4 weeks to wistar rats.controls received an equivalent amount of saline.the animals were sacrificed on days 7 , 14 , 21 and 28 . the body weight and liver weight of rats in earch group were measure .the changes of hepatic stellate (HSCs)、hepatic endothelial cell、hepatic kupffer cell、hepatic pit cell、 hepatocyte、Serum Hyaluronic Acid (HA)、 serum typeIV collagen and Laminin (LN) in different periods of LF were observed variation using light microscope、electron microscope、Immunohistochemical 、Histochemical methods. The result were statistically evaluated using spss10.0<WP=7>analysis .The results: An increase in liver weight was observed by the 7th of DMN treatment , with a decreased liver weight by the 28th days .The maximum liver and body weight ratio increased on the 14th day , but it the decrease on 28st day . Light and electron microscopy: DMN-tread rats showed evidence of hemorrhagic necrosis, mainly centrolobular was evident on the 7day , showing disruption of sinusoidal lining and disappearance of the space of Disse invaded by and large and small mononuclelar cells , the extent of parenchymal damage was characterized under the election microscope and included alterations of the sinusoidal wall with formation of large gaps between endothelial cells and enlarged perisinusoidal paces containing erythrocytes orhrombocytes ocasional ’Pit cells’.plenty of liver kupffer cells appeared in the period and enhanced the ability of releasing inflammatory factor.with time, fibrotic septa were associated with cells bearing transitional features between those of hepatic stellate cells , myofibroblasts and fibroblasts , some pit cells apparently gained access into the space of Disse through large openings of the endothelial wall and were found closely apposed to the hepatocytes, all rats showed micronodular fibrosis in the hepatic parenchyma , electron microscopy showed alterations of the sinusoidal wall the presence of numerous erythrocytes in the spaces of Disse.At day 28 , there was observed in all rats similar findings in the hepatic parenchyma, under the electron microscope, fibrous septa contain abundant collagen bundles , extended segment of the sinusoidal wall were still deprived of endothelium and fibrous septa contained large myofibroblasts and fibroblast , sinusoidal capilarization.Immunohistochemical stain method : For the simultaneous detection of alpha smooth muscle actin(a-SMA),as a maker of activated Hepatic stellate cells<WP=8>(HSCs) in controls, a-SMA positive cells were detected mainly in the portal space either as elements of vascular walls or as fibroblast-like cells scattered in the connective tissue or finely apposed to bile ductudes.A significant increase of a-SMA positive cells was observed already at 7thday after the DMN injection.these cells showed an elongated fibroblast-like shape and.their number progressively increased throughout the experiments,reaching a maximum at 28th day using five-grade classified statistics to analyse the extent of hepatic fibrosis ,there was significant difference between control group and DMN groups(P<0.05) betweenDMN groups there were significant difference too. Histochemical stain method : Tiss更多还原