A Role of Protein Tyrosine Phosphorylation in Bile Ductular Metaplasia of Hepatocytes

已知慢性肝病伴门静脉纤维化和炎症（胆管反应）时，门静脉区胆管增加。新形成的胆管可能来源于门静脉周围肝细胞，但其机制尚不清楚。在这项研究中，我们利用三维培养系统研究了蛋白酪氨酸磷酸化在肝细胞胆管化生中的可能作用。我们还研究了肝非实质细胞，特别是库普弗细胞是否影响肝细胞的分化。我们的研究结果总结如下。(1)将成熟大鼠肝细胞的蛇形聚集体包埋在三维胶原凝胶基质中，树突状细胞突起从聚集体中延伸出来，这些突起表达胆管特异性细胞角蛋白，如细胞角蛋白19和20。体外培养3周后，这些细胞过程形成胆管样管状结构，具有明显的基底膜。(2)原钒酸钠是一种原型蛋白酪氨酸磷酸酶抑制剂，可提高细胞蛋白蛋白酪氨酸磷酸化水平，促进培养的肝细胞树突状形态发生和胆管特异性细胞角蛋白的表达。(3)来源于肝非实质细胞的可溶性因子，特别是已知由库普弗细胞分泌的白细胞介素6和转化生长因子α，显著增强了培养肝细胞的树突状形态发生。我们的数据表明，胶原基质内成熟肝细胞的胆管分化可能受到蛋白酪氨酸磷酸化的调节，并受到非实质细胞源性可溶性因子的影响。

Bile ductules are known to increase in the portal area in chronic liver diseases with portal fibrosis and inflammation (ductular reaction). It has been considered that newly formed bile ductules may be derived from periportal hepatocytes, although the mechanism is not clear. In this study, we investigated a possible role of protein tyrosine phosphorylation in the bile ductular metaplasia of hepatocytes using a three dimensional culture system. We also studied whether liver non-parenchymal cells, especially Kupffer cells, affected hepatocyte differentiation. Summary of our results are as follows.(1) When sheroidal aggregates of mature rat hepatocytes were embedded within a three-dimensional collagen gel matrix, dendritic cellular processes elongated from the aggregates and these processes expressed bile duct-specific cytokeratins, such as cytokeratins 19 and 20. These cellular processes formed bile duct-like tubular structures with distinct basal membrane after 3 weeks in vitro.(2) A prototype protein tyrosine phosphatase inhibitor, sodium orthovanadate, which increased protein tyrosine phosphorylation levels of cellular proteins, promoted the dendritic morphogenesis and expression of the bile duct-specific cytokeratins of cultured hepatocytes.(3) The dendritic morphogenesis of cultured hepatocytes was markedly enhanced by soluble factors derived from liver non-parenchymal cells, especially interleukin 6 and transforming growth factor α, which are known to be secreted by Kupffer cells.Our data suggested that bile ductular differentiation of mature hepatocytes within the collagenous matrix might be regulated by protein tyrosine phosphorylation and affected by non-parenchymal cell-derived soluble factors.