Desmoulière A, Darby I, Costa A M, Raccurt M, Tuchweber B, Sommer P, Gabbiani G
Centre National de la Recherche Scientifique, Institut de Biologie et Chimie des Protéines, Lyon, France.
Lab Invest. 1997 Jun;76(6):765-78.
Early studies showed that during hepatic fibrosis induced by bile duct ligation, fibroblasts within the portal tracts proliferate and express alpha-smooth muscle (SM) actin, suggesting that they may be involved in the deposition of extracellular matrix components in cholestatic fibrosis. Thus, we investigated the deposition of extracellular matrix components (laminin, fibronectin EIIIA, collagen I and IV, procollagen III, elastin, tenascin) as well as the expression of lysyl oxidase and of alpha-SM actin in the portal zone at 24, 48, and 72 hours and 7 days after ligation of the common bile duct. Rat liver tissues were processed for immunofluorescence, in situ hybridization, immunohistochemistry, and for electron and immunoelectron microscopy. At all times examined after bile duct ligation, laminin was observed essentially in the basal membrane of vessels and portal ductules. In sham-operated animals, the fibronectin EIIIA was present exclusively in vessels; at 24 hours postinjury, fibronectin EIIIA expression appeared in both the portal zone and along sinusoids. Two days after ligation, increased expressions of collagen I and IV, procollagen III, and elastin were observed within the portal zone, compared with sham-operated animals. The deposition of these components increased thereafter. Tenascin expression increased soon after bile duct ligation in stroma surrounding proliferating ductules, reaching a maximum at 48 hours; thereafter, expression was restricted to the periphery of proliferating ductules. By in situ hybridization, procollagen I and tissue inhibitor of metalloproteinase-1 mRNA expression was greatly increased in periductular areas at 24 hours postligation and remained elevated throughout the experiment. At 24 hours, a strong reactivity for lysyl oxidase appeared in the portal zone, and, as in controls, alpha-SM actin expression was restricted to vascular SM cells. In the stroma adjacent to proliferating ductules, alpha-SM actin appeared at 48 hours, and the number of alpha-SM actin-positive cells increased until the 7th day. Lysyl oxidase staining increased until 72 hours after bile duct ligation, when it was located in areas surrounding the myofibroblastic cells. At 7 days, lysyl oxidase expression was restricted around myofibroblastic cells present at the periphery of the reactive tissue and appeared to extend into the surrounding parenchyma. These results show that after bile duct ligation, extracellular matrix deposition, and lysyl oxidase expression occur very early in portal connective tissue surrounding proliferating ductules, and precede myofibroblastic differentiation, ie, alpha-SM actin expression. In addition, the data are compatible with the suggestion that in the bile duct ligation model, myofibroblastic differentiation represents an adaptive response to modification of the extracellular matrix environment.
早期研究表明，在胆管结扎诱导的肝纤维化过程中，门管区内的成纤维细胞增殖并表达α-平滑肌（SM）肌动蛋白，提示它们可能参与胆汁淤积性纤维化中细胞外基质成分的沉积。因此，我们研究了胆总管结扎后24、48、72小时及7天时，门管区细胞外基质成分（层粘连蛋白、纤连蛋白EIIIA、胶原蛋白I和IV、前胶原蛋白III、弹性蛋白、腱生蛋白）的沉积以及赖氨酰氧化酶和α-SM肌动蛋白的表达。对大鼠肝组织进行免疫荧光、原位杂交、免疫组织化学以及电子和免疫电子显微镜检查。在胆管结扎后的所有检查时间点，层粘连蛋白主要在血管和门管小叶的基底膜中观察到。在假手术动物中，纤连蛋白EIIIA仅存在于血管中；损伤后24小时，纤连蛋白EIIIA表达出现在门管区和沿肝血窦处。结扎后两天，与假手术动物相比，门管区内胶原蛋白I和IV、前胶原蛋白III和弹性蛋白的表达增加。此后这些成分的沉积增加。腱生蛋白表达在胆管结扎后不久在增生的小叶周围基质中增加，在48小时达到最大值；此后，表达局限于增生小叶的周边。通过原位杂交，结扎后24小时，前胶原蛋白I和金属蛋白酶组织抑制剂-1 mRNA表达在胆管周围区域大幅增加，并在整个实验过程中保持升高。24小时时，门管区出现强烈的赖氨酰氧化酶反应性，与对照组一样，α-SM肌动蛋白表达局限于血管平滑肌细胞。在增生小叶相邻的基质中，α-SM肌动蛋白在48小时出现，α-SM肌动蛋白阳性细胞数量增加直至第7天。胆管结扎后直到72小时赖氨酰氧化酶染色增加，此时它位于肌成纤维细胞周围区域。7天时，赖氨酰氧化酶表达局限于反应性组织周边的肌成纤维细胞周围，并似乎延伸至周围实质。这些结果表明，胆管结扎后，细胞外基质沉积和赖氨酰氧化酶表达在增生小叶周围的门管结缔组织中很早就发生，并先于肌成纤维细胞分化，即α-SM肌动蛋白表达。此外，这些数据与以下观点一致，即在胆管结扎模型中，肌成纤维细胞分化代表对细胞外基质环境改变的一种适应性反应。
