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目的探讨肝坏死后再生过程澡肝窦内皮细胞的形态学变化及其再生机制。方法Wistar雄性大白鼠60只,其中实验组为50只,非处理组及生理盐水组各5只作为对照组。实验组分10个小组,在一次性注射二甲基亚硝胺(DMN)50 mg/kg后,分别于第12、24、36小时和第2、3、5、7、8、10及14天乙醚麻醉下处死,迅速取肝组织、骨髓及外周血。采用HE、免疫组织化学、双重免疫荧光标记方法通过光镜、电镜予以观察。结果注射DMN后12 h肝组织内开始出现小灶状坏死,第24小时逐渐明显。第36小时坏死最明显,并且坏死灶内浸润大量的ED-1(大白鼠单核细胞/吞噬细胞标记物)阳性细胞。注射后第2天和第3天,坏死组织碎片和红细胞被ED-1阳性的吞噬细胞吞噬消除。第5天,在肝组织坏死灶内一些单核细胞其形念由圆形转变成梭形。第7天,细胞与残存的网织纤维接触,并表达SE-1(大白鼠肝窦内皮细胞标记物)及Tie-1(内皮细胞特异性受体),此时常见ED-1/SE-1及ED-1/Tie-1双重阳性的梭形细胞。第8天,除了病变范围变小外,其肝组织形态相似于第7天。到第10天,增生的肝组织数量增加,充填坏死区。第14天坏死组织几乎完全被再生的肝及纤维组织所取代。注射DMN后12 h骨髓中ED-1阳性的单核细胞开始增生,其中部分细胞表达5-溴-2-脱氧尿苷(BrdU)/ED-1,并在36 h其数量达高峰。而这些细胞形态相似于骨髓中圆形单核细胞,其在第24小时至第10天出现于外周血中,高峰时间与骨髓相同。外周血中这些细胞的形状与肝组织坏死区内的ED-1阳性细胞相似。结论注射DMN后圆形Ed-1阳性单核细胞首先在骨髓内增殖,通过血液循环到达肝坏死区域,再分化为肝窦内皮细胞,即DMN介导的坏死区域的肝窦重建可能部分通过血管新生来完成。
Objective To investigate the morphological changes of endothelial cells of the liver cirrhosis and its mechanism of regeneration after hepatic necrosis. Methods Sixty male Wistar rats were randomly divided into experimental group (n = 50), untreated group (n = 5) and saline group (n = 5). The experimental group consisted of 10 subgroups: DMN (50 mg / kg) given at the 12th, 24th, 36th hour and 2nd, 3rd, 5th, 7th, 8th, 10th and 14th Diethyl ether anesthesia was executed quickly to take liver tissue, bone marrow and peripheral blood. HE, immunohistochemistry, double immunofluorescence labeling method by light microscopy, electron microscopy to be observed. Results At the 12th hour after injection of DMN, small focal necrosis began to appear in the liver tissue and gradually became obvious at the 24th hour. Necrosis was most evident at 36 hours, and a significant amount of ED-1 (mouse white blood cell / phagocyte marker) positive cells were infiltrated into the necrotic lesion. On day 2 and 3 after injection, necrotic tissue fragments and erythrocytes were phagocytosed by ED-1-positive phagocytes. On day 5, some of the mononuclear cells in the necrotic lesions of liver tissue changed their shape from round to fusiform. On the 7th day, the cells contacted the remaining reticular fibers and expressed SE-1 (a marker for hepatic sinusoidal endothelial cells of the rat) and Tie-1 (endothelial-cell-specific receptor) 1 and ED-1 / Tie-1 double positive spindle cells. On the 8th day, the hepatic morphology was similar to that on the 7th day, except the lesion was smaller. By day 10, the number of hyperplastic liver tissues increased, filling the necrotic area. Day 14 Necrotic tissue was almost completely replaced by regenerated liver and fibrous tissue. ED-1-positive mononuclear cells in the bone marrow started to proliferate 12h after injection of DMN, and some of them expressed BrdU / ED-1 and peaked at 36 h. These cells are morphologically similar to round mononuclear cells in the bone marrow that appear in peripheral blood from the 24th hour to the 10th day at the same time as the bone marrow. The shape of these cells in peripheral blood is similar to ED-1 positive cells in the necrotic area of liver tissue. Conclusion Circular Ed-1 positive mononuclear cells (DMEM) proliferate first in bone marrow after injection of DMN, reach the area of hepatic necrosis through blood circulation and re-differentiate into hepatic sinusoidal endothelial cells, that is, DMN-mediated necrosis of the sinusoid may partially through the blood vessels Newborn to complete.