Auth Marcus-K H, Boost Kim A, Leckel Kerstin, Beecken Wolf-Dietrich, Engl Tobias, Jonas Dietger, Oppermann Elsie, Hilgard Philip, Markus Bernd H, Bechstein Wolf-Otto, Blaheta Roman A
Universitäts-Kinderklinik Essen,Abteilung für Allgemeine Pädiatrie, Germany.
World J Gastroenterol. 2005 Apr 14;11(14):2080-7. doi: 10.3748/wjg.v11.i14.2080.
Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC.
Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting.
In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel.
Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome de-differentiation, which occurs during continuous stimulation by means of growth factors.
体外培养的人肝细胞（HC）生长和分化能力的逐渐丧失阻碍了其临床应用。本研究旨在评估双相培养技术对成人HC生长因子受体表达与激活及分化的影响。
分离得到的HC先后在富含激素的分化培养基（DM）中培养，该培养基含有烟酰胺、胰岛素、转铁蛋白、硒和地塞米松，或在激活培养基（AM）中培养，该培养基含有肝细胞生长因子（HGF）、表皮生长因子（EGF）和粒细胞巨噬细胞集落刺激因子（GM-CSF）。通过荧光测定法、共聚焦显微镜和蛋白质印迹法检测HC受体（MET和EGFR）的表达、分布和激活情况以及特征性细胞角蛋白（CK）丝的模式。
在双相培养系统中，HC经历了反复的激活周期（以生长因子受体的表达和激活为特征）和再分化（以典型的CK-18丝分布但CK-19表达低或无表达为特征）。在AM中，MET和EGFR表达增加与受体转位到细胞质以及非典型CK-19的诱导有关。在DM中，MET和EGFR低表达定位于细胞膜，CK-19减少。受体磷酸化需要将HC包埋在I型胶原凝胶中。
当提供来自细胞外基质的特定信号和连续的生长刺激时，体外可实现对成熟人HC生长因子受体激活的控制和可逆调节。双相技术有助于克服因生长因子持续刺激而发生的去分化。
