Objective To seek an appropriate culture condition in which porcine hepatocytes can continuously proliferate and express their normal differentiation phenotypes for a longer period of time. Methods Different types a...Objective To seek an appropriate culture condition in which porcine hepatocytes can continuously proliferate and express their normal differentiation phenotypes for a longer period of time. Methods Different types and dosages of reagents,transferrin,epidermal growth factor and nicotinamide were used to culture porcine hepatocytes in serum-free Dulbecco’s modified Eagle’s medium (DMEM). And the proliferative effects and functions of the cells were detected at different culture times. Results Transferrin at 5 ng/ml,nicotinamide at 10 mmol/L and epidermal growth factor at 10 ng/ml had better effects on the viability of hepatocytes than DMEM,DMEM+10% fetal cattle serum FCS and other dosages of these reagents ( P <0.05). OD values of MTS were still high in culture at day 7 and day 10,while nearly 30%-35% cells went into the S phase. Good hepatocyte funcitons were found in these groups,and the secretion of albumin was positively correlated with OD value of MTS. The levels of aspartate transaminase and ammonia in these media were lower than those in DMEM and DMEM+FCS.Conclusion Transferrin at 5 ng/ml,epidermal growth factor at 10 ng/ml and nicotinamide at 10 mmol/L are benefitial to the viability and proliferation of hepatocytes.展开更多
AIM： To study the effect of IL-10 on the expression of growth factors - transforming growth factor-β1 （TGF-β1）, epidermal growth factor （EGF）, hepatocyte growth factor （HGF）and platelet-derived growth factor ...AIM： To study the effect of IL-10 on the expression of growth factors - transforming growth factor-β1 （TGF-β1）, epidermal growth factor （EGF）, hepatocyte growth factor （HGF）and platelet-derived growth factor （PDGF） of hepatic stellate cells （HSCs） of hepatic fibrosis rat and the anti-fibrogenic role of exogenous IL-10. METHODS： Hepatic fibrosis was induced by CCI4 administration intra-peritoneally. Sixty clean male Sprague-Dawley （SD） rats were randomly divided into three groups： normal control group （GN, 8 rats）, hepatic fibrosis model group （GC, 28 rats） and IL-10 treated group （GI, 24 rats）. At the beginning of the 7^th and 11^th wk, rats in each group were routinely perfused with pronase E and type IV collagenase through a portal vein catheter and the suspension obtained from the liver was spun by centrifugation with 11% Nycodenz density gradient to isolate HSCs. Histological examination was used to determine the degree of hepatic fibrosis. RT-PCR was employed to analyze mRNA expression from freshly isolated cells. Immunocytochemistry was performed to detect protein expression in primary cultured HSCs. RESULTS： Rat hepatic fibrosis was developed with the increase of injection frequency of CCl4, and HSCs were successfully isolated. At the 7^th and 11^th wk, TGF-β1, EGF, and HGF mRNA in GC increased obviously compared with GN （P = 0.001/0.042, 0.001/0.001, 0.001/0.001） and GI （P= 0.001/0.007, 0.002/0.001, 0.001/0.001）. For TGF-β1, no difference was observed between GI and GN. For EGF, mRNA level in GI increased compared with GN during the 7^th wk （P= 0.005） and 11^th wk （P= 0.049）. For HGF, mRNA level in GI decreased compared with GN at the 7^th wk （P = 0.001） and 11^th wk （P= 0.021）. Between these two time points, TGF-β1 expression at the 7^th wk was higher than that of the 11^th wk （P = 0.049）, but for EGF, the former was lower than the latter （P = 0.022）. As for PDGF mRNA, there was no significant difference between thesegroups, but difference seemed to exist in protein levels. Results by immunocytochemistry of TGF-β1 and EGF were paralleled with the above findings. CONCLUSION： The expression of TGF-β1, EGF and HGF increased in HSC of hepatic fibrosis rat and decreased after treatment with IL-10. IL-10 plays an anti-fibrogenic role by suppressing growth factors expression.展开更多
Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the cont...Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the contribution of hepatic progenitors becomes very relevant. Here, we present an update of recent studies on growth factors and cytokine-driven intracellular pathways that govern liver stem/pro-genitor cell expansion and differentiation, and the rel-evance of these signals in liver development, regeneration and carcinogenesis. Tyrosine kinase receptor signaling, in particular, c-Met, epidermal growth factor receptors or fibroblast growth factor receptors, contribute to prolifera-tion, survival and differentiation of liver stem/progenitor cells. Different evidence suggests a dual role for the trans-forming growth factor (TGF)-β signaling pathway in liver stemness and differentiation. On the one hand, TGF-βmediates progression of differentiation from a progenitor stage, but on the other hand, it contributes to the expan-sion of liver stem cells. Hedgehog family ligands are nec-essary to promote hepatoblast proliferation but need to be shut off to permit subsequent hepatoblast differentiation. In the same line, the Wnt family and β-catenin/T-cell fac-tor pathway is clearly involved in the maintenance of liver stemness phenotype, and its repression is necessary for liver differentiation during development. Collectively, data indicate that liver stem/progenitor cells follow their own rules and regulations. The same signals that are essential for their activation, expansion and differentiation are good candidates to contribute, under adequate conditions, to the paradigm of transformation from a pro-regenerative to a pro-tumorigenic role. From a clinical perspective, this is a fundamental issue for liver stem/progenitor cell-based therapies.展开更多
AIM: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 expr...AIM: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. METHODS: 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 (GMCSF). 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. RESULTS: 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. CONCLUSION: 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 dedifferentiation, which occurs during continuous stimulation by means of growth factors.展开更多
文摘Objective To seek an appropriate culture condition in which porcine hepatocytes can continuously proliferate and express their normal differentiation phenotypes for a longer period of time. Methods Different types and dosages of reagents,transferrin,epidermal growth factor and nicotinamide were used to culture porcine hepatocytes in serum-free Dulbecco’s modified Eagle’s medium (DMEM). And the proliferative effects and functions of the cells were detected at different culture times. Results Transferrin at 5 ng/ml,nicotinamide at 10 mmol/L and epidermal growth factor at 10 ng/ml had better effects on the viability of hepatocytes than DMEM,DMEM+10% fetal cattle serum FCS and other dosages of these reagents ( P <0.05). OD values of MTS were still high in culture at day 7 and day 10,while nearly 30%-35% cells went into the S phase. Good hepatocyte funcitons were found in these groups,and the secretion of albumin was positively correlated with OD value of MTS. The levels of aspartate transaminase and ammonia in these media were lower than those in DMEM and DMEM+FCS.Conclusion Transferrin at 5 ng/ml,epidermal growth factor at 10 ng/ml and nicotinamide at 10 mmol/L are benefitial to the viability and proliferation of hepatocytes.
基金Supported by Technology and Science Foundation of Fujian Province, No. 2003 D05
文摘AIM： To study the effect of IL-10 on the expression of growth factors - transforming growth factor-β1 （TGF-β1）, epidermal growth factor （EGF）, hepatocyte growth factor （HGF）and platelet-derived growth factor （PDGF） of hepatic stellate cells （HSCs） of hepatic fibrosis rat and the anti-fibrogenic role of exogenous IL-10. METHODS： Hepatic fibrosis was induced by CCI4 administration intra-peritoneally. Sixty clean male Sprague-Dawley （SD） rats were randomly divided into three groups： normal control group （GN, 8 rats）, hepatic fibrosis model group （GC, 28 rats） and IL-10 treated group （GI, 24 rats）. At the beginning of the 7^th and 11^th wk, rats in each group were routinely perfused with pronase E and type IV collagenase through a portal vein catheter and the suspension obtained from the liver was spun by centrifugation with 11% Nycodenz density gradient to isolate HSCs. Histological examination was used to determine the degree of hepatic fibrosis. RT-PCR was employed to analyze mRNA expression from freshly isolated cells. Immunocytochemistry was performed to detect protein expression in primary cultured HSCs. RESULTS： Rat hepatic fibrosis was developed with the increase of injection frequency of CCl4, and HSCs were successfully isolated. At the 7^th and 11^th wk, TGF-β1, EGF, and HGF mRNA in GC increased obviously compared with GN （P = 0.001/0.042, 0.001/0.001, 0.001/0.001） and GI （P= 0.001/0.007, 0.002/0.001, 0.001/0.001）. For TGF-β1, no difference was observed between GI and GN. For EGF, mRNA level in GI increased compared with GN during the 7^th wk （P= 0.005） and 11^th wk （P= 0.049）. For HGF, mRNA level in GI decreased compared with GN at the 7^th wk （P = 0.001） and 11^th wk （P= 0.021）. Between these two time points, TGF-β1 expression at the 7^th wk was higher than that of the 11^th wk （P = 0.049）, but for EGF, the former was lower than the latter （P = 0.022）. As for PDGF mRNA, there was no significant difference between thesegroups, but difference seemed to exist in protein levels. Results by immunocytochemistry of TGF-β1 and EGF were paralleled with the above findings. CONCLUSION： The expression of TGF-β1, EGF and HGF increased in HSC of hepatic fibrosis rat and decreased after treatment with IL-10. IL-10 plays an anti-fibrogenic role by suppressing growth factors expression.
基金Supported by Grants from the Ministerio de Ciencia e Innovación, MICINN, Spain (SAF2009-12477 to Sánchez A BFU2009-07219 and ISCIII-RTICC RD06/0020 to Fabregat I)+1 种基金AGAUR-Generalitat de Catalunya (2009SGR-312 to Fabregat I)UCM-BSCH (920359 to Sánchez A)
文摘Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the contribution of hepatic progenitors becomes very relevant. Here, we present an update of recent studies on growth factors and cytokine-driven intracellular pathways that govern liver stem/pro-genitor cell expansion and differentiation, and the rel-evance of these signals in liver development, regeneration and carcinogenesis. Tyrosine kinase receptor signaling, in particular, c-Met, epidermal growth factor receptors or fibroblast growth factor receptors, contribute to prolifera-tion, survival and differentiation of liver stem/progenitor cells. Different evidence suggests a dual role for the trans-forming growth factor (TGF)-β signaling pathway in liver stemness and differentiation. On the one hand, TGF-βmediates progression of differentiation from a progenitor stage, but on the other hand, it contributes to the expan-sion of liver stem cells. Hedgehog family ligands are nec-essary to promote hepatoblast proliferation but need to be shut off to permit subsequent hepatoblast differentiation. In the same line, the Wnt family and β-catenin/T-cell fac-tor pathway is clearly involved in the maintenance of liver stemness phenotype, and its repression is necessary for liver differentiation during development. Collectively, data indicate that liver stem/progenitor cells follow their own rules and regulations. The same signals that are essential for their activation, expansion and differentiation are good candidates to contribute, under adequate conditions, to the paradigm of transformation from a pro-regenerative to a pro-tumorigenic role. From a clinical perspective, this is a fundamental issue for liver stem/progenitor cell-based therapies.
基金Supported by the "Matthias Lackas-Stiftung", "Paul und Ursula Klein-Stiftung", "Heinrich und Erna Schaufler-Stiftung", "Gisela Stadelmann-Stiftung", and study grants from the Johann Wolfgang Goethe-Universitatsklinikum,Universitatsklinikum Essen (IFORES),and Deutsche Forschungsgemeinschaft (AU 117/4-1)
文摘AIM: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. METHODS: 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 (GMCSF). 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. RESULTS: 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. CONCLUSION: 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 dedifferentiation, which occurs during continuous stimulation by means of growth factors.
