Michalopoulos G K, Bowen W, Nussler A K, Becich M J, Howard T A
Department of Pathology, University of Pittsburgh, Pennsylvania 15261.
J Cell Physiol. 1993 Sep;156(3):443-52. doi: 10.1002/jcp.1041560303.
Hepatocytes maintained in collagen gels remain differentiated for prolonged periods of time compared to cells maintained on conventional cultures. Previous studies with other culture systems in which chemical supplements or substratum modifications enhanced hepatocyte differentiation showed that in all of these systems hepatocytes do not respond to mitogens. In this study it is shown that hepatocytes maintained between two layers of collagen gels respond to mitogens HGF (also known as scatter factor (HGF/SF)) and epidermal growth factor (EGF). Cell density did not affect the responsiveness to mitogens as in conventional cultures. In addition both mitogens (HGF more pronounced) induce characteristic morphogenic changes in which hepatocytes form processes and join in formation of cords. Hepatocytes respond to mitogens for up to 6 days in culture at which point they become refractory to further mitogenic stimulation. This occurs despite electron microscopic evidence that these cells are fully viable when they become refractory to mitogenesis. The refractory state is not modified by substitution of one growth factor for the other or by addition of growth factors at different times. Hepatocytes in the refractory state become again responsive to mitogens when the collagen gels are dispersed by collagenase and the cells are replated on conventional substrates.
与在传统培养条件下培养的细胞相比，在胶原凝胶中培养的肝细胞能在更长时间内保持分化状态。以往对其他培养系统的研究表明，在这些系统中，化学添加剂或基质修饰可增强肝细胞分化，但肝细胞对促有丝分裂原均无反应。本研究表明，夹在两层胶原凝胶之间培养的肝细胞对促有丝分裂原肝细胞生长因子（也称为分散因子（HGF/SF））和表皮生长因子（EGF）有反应。与传统培养不同，细胞密度并不影响对促有丝分裂原的反应性。此外，两种促有丝分裂原（肝细胞生长因子的作用更显著）均可诱导特征性的形态发生变化，即肝细胞伸出突起并形成索状结构。在培养过程中，肝细胞对促有丝分裂原的反应可持续6天，此后它们对进一步的促有丝分裂刺激不再敏感。尽管电镜证据显示，这些细胞在对有丝分裂原产生不应性时仍具有完全的活力，但这种不应性状态依然会出现。用一种生长因子替代另一种生长因子，或在不同时间添加生长因子，均不能改变这种不应性状态。当用胶原酶分散胶原凝胶，将细胞重新接种到传统基质上时，处于不应性状态的肝细胞会再次对促有丝分裂原产生反应。
