The study of cholestasis using immunoelectron microscopy and rapid freezing deep-etching replica

采用免疫荧光、透射电镜（TEM）和免疫电镜（IEM）观察了各种胆汁淤积模型大鼠肝细胞的细胞骨架变化。以大鼠肝内胆汁淤积模型和梗阻性黄疸模型分别以phalloidin、cytokalasin B和胆汁淤积因子治疗肝内胆汁淤积模型。用Katsuma等人的方法处理的肝组织中，透射电镜（TEM）可以清楚地显示出管周鞘（PS）。几乎所有组成PS的细丝都被IEM鉴定为中间细丝。大鼠经phalloidin治疗后胆管结扎后，微丝（microfilament， MFS）和IFs数量增加，PS扩张增厚。胆管结扎大鼠出现局灶性PS破坏，胆管再通后消失。这些结果表明，在胆汁淤积症中，IFs的数量随着MFs的增加而增加。我们还利用几种单克隆抗细胞角蛋白（CK）抗体No.8和No. 18（Moll分类）研究了肝细胞中IFs的免疫异质性，并在肝癌发生实验模型的增生性结节中观察到肝细胞IFs的变化。免疫荧光和免疫电镜显示，细胞角蛋白多肽的染色模式在肝癌发生过程中发生了变化，这可能有助于肝癌的早期发现。

We investigated the cytoskeletal changes of hepatocytes in various kinds of cholestatic rat models using immunofluorescence, transmission electron microscope(TEM) and immunoelectron microscope(IEM). The rats treated with phalloidin, cytokalasin B and cholestatic factor were used as the model of in- trahepatic cholestasis and the rats as the model of obstructive jaundice.Pericanalicular sheath(PS) was clearly demonstrated by TEM in the liver tissues treated by the method of Katsuma et al. Almost all filaments made up PS were identified as intermediate filaments(IFs) by IEM.In the phalloidin treated and bile duct ligated rats, microfilaments(MFS) and IFs increased in amounts and PS was dilated and thickened. The focal disruption of the PS was observed in the bile duct ligated rats, which disappeared after recanalization of the bile ducts. These results suggest that IFs increase in amounts according to the increase of MFs in cholestasis.We also investigated the immunological hetetogeneity of IFs in hepatocytes using a few kinds of monoclonal anti cytokeratin(CK) antibodies including No.8 and No. 18(Moll's classification) and the change of IFs of hepatocytes was noted in the hyperplastic nodules in the experimental model of hepatocacinogenesis. Immunofluorescence and immunoelectron microscopy demonstrated that the staining pattern of cytokeratin polypeptides changed in the process of hepatocarcinogenesis, which might be useful in the early detection of hepatocellular carcinoma.