实验性肝坏死后再生肝窦内皮细胞的形态学观察及机制探讨

目的探讨肝坏死后再生过程中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）50mg／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12h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J彬吞噬细胞标记物）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态由圆形转变成梭形。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12h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36h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。

Morphologic observation and pathogenesis investigation of regenerated sinusoidal endothelial cells in remodelling rat hepatic necrotic tissue

OBJECTIVE: To investigate the morphological changes and regeneration mechanism of sinusoidal endothelial cell. METHODS: Sixty male Wistar rats (bought from SLC company limited of Japan) were divided into three groups. Fifty of them belonged to experiment group, five rats belonged to untreated group, and the rest five ones belonged to normal saline treated group. The experiment group was then divided into ten subgroups. All the rats of the experiment group were killed under anaesthesia using aether at 12, 24, 36 hrs, and 2, 3, 5, 7, 8, 10 and 14 days subsequently after an one-off injection of dimethylnitrosamine (DMN) (50 mg/kg). The liver tissues, bone marrows and peripheral blood of the rats were taken out rapidly. All the tissues received with HE staining, immunohistochemistry staining and double immunofluorescence labelings, and they were observed under a light microscope and electron microscope. The livers, bone marrows and peripheral blood from the rats at 24 hrs to 14 days after an injection of DMN were examined by light microscopic, immunohistochemical, and ultrastructural methods. RESULTS: Small focal necrosis of the liver tissues was found at 12 hrs after the DMN injection, and gradually becomes more obvious from the 24 hrs. The most obvious necrosis, with lots of ED-1 (monocyte/phagocyte marker of rats) positive cells infiltration, was observed at 36 hrs. On the 2nd day and 3rd day after injection, the necrotic fragments and red cells were phagocyted by ED-1 positive macrophages. On the 5th day, some of the ED-1-positive cells were transformed from round to spindle in shape. On the 7th day, these cells contacted with residual reticulin fibers and became positive for SE-1, a marker of hepatic sinusoidal endothelial cells and Tie-1, an endothelial cell-specific surface receptor, associated with frequent occurrence of ED-1/SE-1 and ED-1/Tie-1 double positive spindle cells. On the 8th day, the histomorphology of liver tissue was similar with that on day 7, except that the range of the lesions had become smaller. On the 10th day, the regeneration of liver tissue increased, filling in the necrosis. On the 14th day, the necrotic tissues were almost replaced by regenerated liver tissues and thin bundles of central-to-central bridging fibrosis. 12 hrs after the DMN injection, bone marrow studies showed an increase in the number of ED-1 positive mononuclear cells, some of which were both BrdU/ED-1 positive. The number of ED-1 positive mononuclear cells reach their highest level at 36 hrs. These cells are morphologically similar to round mononuclear cells in bone marrows and could be found in the peripheral blood from 24 hrs to the 10 days. They reached their highest level in peripheral blood at the same time as in the bone marrow. These cells morphologically resembled ED-1 positive cells in necrotic tissues of the liver. CONCLUSIONS: These findings suggest that round mononuclear ED-1-positive cells proliferate first in the bone marrow after DMN treatment, reach necrotic areas of livers through circulation, and differentiate to sinusoidal endothelial cells. Namely, hepatic sinusoids in DMN-induced necrotic areas may partly be reorganized possibly by vasculogenesis.