Studies on pathogenesis and progression of primary biliary cirrhosis with special reference to analysis of autoimmune responses to biliary tract cytoskeleton.

透射电子显微镜显示，中间丝（IF）广泛存在于胆管上皮细胞质中，而微丝（肌动蛋白丝：AF）位于质膜下区域。采用间接免疫荧光法，在所有原发性胆汁性肝硬化（PBC）患者血清中均检测到PtK_2培养细胞中IF特异性免疫荧光证实的抗IF（细胞角蛋白：CK）抗体和_3T_3培养细胞中AF特异性免疫荧光证实的抗AF抗体。通过SDS-PAGE将PtK_2细胞来源的CK分为CK1(52kD)和CK2(45kD)亚类。分别产生针对CK1和CK2的单克隆抗体。通过夹心抑制ELISA，证明PBC患者血清中抗CK1抗体滴度显着高于其他肝病患者，在PBC的I-II期中程度最高，肝活检显示慢性非化脓性破坏性胆管炎（CNSDC）最显着的形态特征，在IV期中程度最低，几乎不显示CNSDC。通过长期熊去氧胆酸（UDCA）。有症状和无症状 PBC 患者血清中抗 CK1 和抗 M2（丙酮酸脱氢酶）抗体滴度显着降低，提示 UDCA 治疗可能影响 PBC 的自身免疫异常。UDCA 治疗抗 CK1 抗体滴度下降，反映 PBC 病理状态的改善。通过间接免疫过氧化物酶抗体法，揭示不仅 HLA-DR，而且细胞间粘附分子 (ICAM)-1 在胆管上皮细胞质膜的外表面表达最强烈。此外，还发现淋巴细胞功能相关抗原（LFA）-1在细胞表面表达的CD4和CD8阳性细胞特别浸润在胆管周围，与ICAM-1阳性胆管上皮细胞直接接触。

Transmission electron microscopy revealed that intermediate filaments (IF) are diffusely present in the cytoplasm of bile duct epithelium, whereas microfilaments (actin filametns : AF) are located in the subplasmalemmal region. Acoording to the indirect immunofluorescent method, anti-IF (cytokeratin : CK) antibody proved by the specific immunofluorescence for IF in PtK_2 cultured cells and anti-AF antibody proved by that for AF in _3T_3 cultured cells were detected in sera of all patients with primary biliary cirrhosis (PBC). CK derived from PtK_2 cells were divided into subclass CK1(52kD) and CK2(45kD) by SDS-PAGE.Monoclonal antibodies were produced against CK1 and CK2 respectively. By the sandwich inhibition ELISA, anti-CK1 antibody titers in sera of PBC patients were proved to be significantly higher than those in patients with other liver disease, demonstrating the highest degrees on the stage I-II of PBC showing the most prominent morphological features of chronic non-suppurative destructive cholangitis (CNSDC) by liver biopsy and the lowest degrees on the stage IV hardly showing CNSDC.By long term ursodeoxycholic acid (UDCA). anti-CK1 and anti-M2 (pyruvate dehydrogenase) antibody titers were significantly reduced in sera of symptomatic and asymptomatic PBC patients, implying that UDCA therapy may affect the autoimmune abnormalities in PBC.A decrease in anti-CK1 antibody titers by UDCA therapy would reflect an improvement in pathological state of PBC.By the indirect immunoperoxidase antibody method, it was revealed that not only HLA-DR but also intercellular adhesiom molecule (ICAM)-1 are most strongly expressed on the outer surfaces of the plasma membranes of bile duct epithelial cells. Furthermore CD4 and CD8-positive cells expressed on cell surface by lymphocyte function-associated antigen(LFA)-1 were found to be infiltrated particularly around bile ducts, making direct contant with the ICAM-1-positive bile duct epithelial cells.