肝窦毛细血管化的形成机制研究

Study on the mechanisms of development of hepatic sinusoidal capillarization

目的 探讨二甲基亚硝胺 (DMN)致大鼠肝纤维化过程中肝窦壁病理变化及其与门脉压力的关系。方法 雄性Wistar大鼠 4 0只用 0 .5 %DMN每周连续 3d共 4周 12次腹腔注射制作大鼠肝纤维化模型 ,分别于造模后 1d、2d、3d、1周、2周、4周、6周、8周作为动态观察时相点 ;分别取 5只模型大鼠和 3只正常大鼠肠系膜前静脉分支插管法测门脉压力 (Ppv) ;免疫组化染色观察肝组织Ⅳ型胶原(ColⅣ )、层粘连蛋白 (LM)和Ⅰ型胶原 (ColⅠ )表达 ;透射电镜观察肝组织超微结构。结果 正常大鼠肝窦壁ColⅣ阳性表达 ;模型组肝窦壁除ColⅣ阳性染色呈现为先弱后强外 ,LM和ColⅠ的沉积均随造模时间的延长而进行性增加 ,4周时表达最为明显 ,电镜下可见肝窦内皮失窗孔和内皮下完整基底膜形成 ;模型组Ppv与肝窦壁LM的表达量呈显著正相关 (P <0 .0 5 )。结论 DMN大鼠肝窦内皮下基底膜是在功能性基底膜破坏的基础上 ,随着LM与新合成ColⅣ沉积以及两者结合的增加。

Objective To study the relationship between the liver sinusoidal pathological changes and portal hypertension during the dimethylnitrosamine (DMN) induced liver fibrosis in rats. Methods The rat liver fibrosis model was established by peritoneal injection of DMN (at a dose of 10 mg/kg, 3 times a week, for 4 weeks) in 40 male Wister mice. In the control group of 24 healthy mice, saline solution was injected peritoneally. The dynamic changes of liver fibrosis were observed at different time points (1, 2, 3 day, 1, 2, 4, 6 and 8 week). The pressure of portal vein (Ppv) was directly measured by the intubation of mesentery anterior vein in 5 mice of the model and 3 of the control. The expressions of type Ⅳ collagen (Col Ⅳ), laminin (LM) and type Ⅰ collagen (Col Ⅰ) were detected by immunohistochemistry. Hepatic ultrastructure was observed by electron microscopy. Results Positive expression of Col Ⅳ was observed in the normal liver sinusoidal walls. The various intensities of positive staining of Col Ⅳ were observed in the liver sinusoidal walls of the fibrosis model. Positive expressions of LM and Col Ⅰ increased, and the strongest positive staining displayed in the 4 week model rats. Meanwhile, the fenestrae in the sinusoidal endothelial cells (SECs) were lost and the basal membrane was formed. There was a remarkable positive correlation between the Ppv and the expressions of LM in the DMN induced rat fibrosis( P <0.05). Conclusions Hepatic sinusoidal capillarization (including the loss of fenestrae in the SECs, the formation of basal membrane and the activation of hepatic stellate cells) is the important pathological basis in development of portal hypertension during the DMN induced liver fibrosis in rats. The formation of sinusoidal endothelial basement membrane may be due to the destruction of functional basement membrane, and the deposition of LM, Col Ⅳ and Col Ⅰ.