Studies on Pathogenesis of Intrahepatic Cholestasis - Analysis of Ca^<++>- Calmodulin-Actomyosin System.

为探讨胆汁分泌受损所致肝内胆汁淤积的发病机制，采用急性肝内胆汁淤积模型和急性肝内胆汁淤积患者的肝活检标本，采用免疫荧光和免疫电子显微镜技术，对肝内胆管细胞骨架系统进行了体内和体外研究。通过对锑酸钾法获得的电子显微照片进行数字图像分析，揭示了胞质内游离钙离子的超微结构分布及其变化。胆小管收缩采用延时摄影进行分析。已有研究表明，胞浆内钙离子、钙离子结合蛋白钙调蛋白和钙离子-镁-三磷酸腺苷酶的存在对肌动蛋白细丝介导的胆小管收缩具有重要的调控作用。这三个因素的缺失导致了胆小管…的紊乱。用单层培养的胆小管细胞系_3T_3和PTK_2，用免疫荧光和免疫过氧化物酶方法检测了原发性胆汁性肝硬变(PBC)患者血清中抗中间丝(IF)和抗肌动蛋白细丝(AF)的含量。而抗IF抗体主要是针对PBC I期来源于胆管上皮管腔部位的AF形成的，II期以后又形成于针对胆管周围的AF。免疫荧光和免疫过氧化物酶染色显示，PBC患者肝内胆管上皮细胞中存在人类白细胞抗原-DR的异常表达。提示细胞毒性T细胞可能识别这种HLA-DR，并与胆管上皮细胞发生反应，导致PBC胆管的破坏。较少

In an attempt to elucidate the pathogenesis of intrahepatic cholestasis due to bile secretory impairment, the cytoskeletal system in the intrahepatic bilary tract was investigated in vivo and in vitro by immunofluorescence and immunoelectron microscopy using an experimental model of acute intrahepatic cholestasis and liver biopsy specimens obtained from patients with acute intrahepatic cholestasis. The ultrastructural distribution of intracytoplamic free calcium ions (Ca^<++>) and its alterations were revealed by digital image analysis of the electron micrographs obtained by the potassium antimonate method. Bile canalicular contractions were analysed by time-lapse cinematography. It has been probed that the existence of intracytoplasmic Ca^<++>, Ca^<++>-binding protein calmodulin, and Ca^<++>-Mg^<++>-ATPase is essential for the control of actin filaments-mediated bile canalicular contractions. The depletion of these three factors resulted in the disturbances of bile canalicular contrac … More tions, i. e. the paralytic dilatation of bile canaliculli, causing stagnation of bile in the canaliclus network leading to intrahepatic cholestasis.Anti-intermediate filaments (IF) and anti-actin filaments (AF) were determined in sera of patients with primary biliary cirrhosis (PBC) by immunofluorescence and immunoperoxidase method using the monolayer-culutured cell lines, _3T_3 and PtK_2. It has been considered that anti-IF antibody is formed against the IF derived from the bile duct epithelium as a result of bile duct destruction, while anti-IF antibody is formed mainly against the AF derived from the luminal site of bile duct epithelium at stage I of PBC and is further formed against the AF from pericanalicular region after the stage II. Immunofluorescence and immunoperoxidase microscopy revealed the aberrant expression of HLA-DR within the intrahepatic bile duct epithelium in the biopsy speciments from patients with PBC. It is strongly suggested that cytotoxic T cells may recognize this HLA-DR and react with the bile duct epithelium, leading to the destruction of bile duct in PBC. Less