RESUMO
Hepatic progenitor cells (HPCs) are a potential cell source for liver cell transplantation but do not function like mature liver cells. We sought an effective and reliable method to induce HPC maturation. An immortalized HP14.5 albumin promoter-driven Gaussian luciferase (ALB-GLuc) cell line was established from HPCs isolated from fetal mouse liver of post coitus day 14.5 mice to investigate the effect of induction factors on ALB promoter. HP14.5 parental cells were cultured in DMEM with different combinations of 2% horse serum (HS), 0.1 µM dexamethasone (DEX), 10 ng/mL hepatic growth factor (HGF), and/or 20 ng/mL fibroblast growth factor 4 (FGF4). Trypan blue and crystal violet staining were used to assess cell proliferation with different induction conditions. Expression of hepatic markers was measured by semi-quantitative RT-PCR, Western blot, and immunofluorescence. Glycogen storage and metabolism were detected by periodic acid-Schiff and indocyanine green (ICG) staining. GLuc activity indicated ALB expression. The combination of 2% HS+0.1 µM Dex+10 ng/mL HGF+20 ng/mL FGF4 induced the highest ALB-GLuc activity. Cell proliferation decreased in 2% HS but increased by adding FGF4. Upon induction, and consistent with hepatocyte development, DLK, AFP, and CK19 expression decreased, while ALB, CK18, and UGT1A expression increased. The maturity markers tyrosine aminotransferase and apolipoprotein B were detected at days 3 and 6 post-induction, respectively. ICG uptake and glycogen synthesis were detectable at day 6 and increased over time. Therefore, we demonstrated that HPCs were induced to differentiate into functional mature hepatocytes in vitro, suggesting that factor-treated HPCs may be further explored as a means of liver cell transplantation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Hepatócitos/citologia , Fígado/citologia , Células-Tronco/efeitos dos fármacos , Animais , Antígenos de Diferenciação/análise , Apolipoproteína B-100 , Apolipoproteínas B/isolamento & purificação , Proliferação de Células , Dexametasona/administração & dosagem , Fatores de Crescimento de Fibroblastos/administração & dosagem , Violeta Genciana , Glicogênio/metabolismo , Fator de Crescimento de Hepatócito/administração & dosagem , Verde de Indocianina/farmacocinética , Camundongos , Cultura Primária de Células/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Azul Tripano , Tirosina Transaminase/isolamento & purificaçãoRESUMO
Hepatic progenitor cells (HPCs) are a potential cell source for liver cell transplantation but do not function like mature liver cells. We sought an effective and reliable method to induce HPC maturation. An immortalized HP14.5 albumin promoter-driven Gaussian luciferase (ALB-GLuc) cell line was established from HPCs isolated from fetal mouse liver of post coitus day 14.5 mice to investigate the effect of induction factors on ALB promoter. HP14.5 parental cells were cultured in DMEM with different combinations of 2% horse serum (HS), 0.1 µM dexamethasone (DEX), 10 ng/mL hepatic growth factor (HGF), and/or 20 ng/mL fibroblast growth factor 4 (FGF4). Trypan blue and crystal violet staining were used to assess cell proliferation with different induction conditions. Expression of hepatic markers was measured by semi-quantitative RT-PCR, Western blot, and immunofluorescence. Glycogen storage and metabolism were detected by periodic acid-Schiff and indocyanine green (ICG) staining. GLuc activity indicated ALB expression. The combination of 2% HS+0.1 µM Dex+10 ng/mL HGF+20 ng/mL FGF4 induced the highest ALB-GLuc activity. Cell proliferation decreased in 2% HS but increased by adding FGF4. Upon induction, and consistent with hepatocyte development, DLK, AFP, and CK19 expression decreased, while ALB, CK18, and UGT1A expression increased. The maturity markers tyrosine aminotransferase and apolipoprotein B were detected at days 3 and 6 post-induction, respectively. ICG uptake and glycogen synthesis were detectable at day 6 and increased over time. Therefore, we demonstrated that HPCs were induced to differentiate into functional mature hepatocytes in vitro, suggesting that factor-treated HPCs may be further explored as a means of liver cell transplantation.
Assuntos
Animais , Camundongos , Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Hepatócitos/citologia , Fígado/citologia , Células-Tronco/efeitos dos fármacos , Antígenos de Diferenciação/análise , Apolipoproteínas B/isolamento & purificação , Proliferação de Células , Dexametasona/administração & dosagem , Fatores de Crescimento de Fibroblastos/administração & dosagem , Violeta Genciana , Glicogênio/metabolismo , Fator de Crescimento de Hepatócito/administração & dosagem , Verde de Indocianina/farmacocinética , Cultura Primária de Células/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Azul Tripano , Tirosina Transaminase/isolamento & purificaçãoRESUMO
OBJECTIVES/HYPOTHESIS: Hepatocyte growth factor (HGF) is a multifunctional polypeptide that plays various roles in embryogenesis and tissue regeneration and exhibits marked antifibrotic activity. The present study sought to assess the effects of HGF injection and reinjection coinciding with its peak of activity on collagen density, vessel density, inflammatory reaction in the lamina propria, and mean epithelial thickness in the injured rabbit vocal fold. STUDY DESIGN: Prospective, controlled, experimental animal study. METHODS: Fourteen rabbits were subdivided into two groups and underwent injury of the vocal folds. Immediately after injury, animals in group 1 received HGF injections into the right vocal fold (RVF), whereas those in group 2 received bilateral HGF injections and a single reinjection into the RVF 10 days after the first, to coincide with the peak of HGF activity. The left vocal folds (LVFs) served as controls in both groups. Histological assessment of laryngeal specimens was performed at 30 and 40 days, respectively. RESULTS: In both groups, collagen density was lower in the right (treated) vocal folds than in the left (control) folds (P=0.018). Vessel density was higher in the RVFs in group 2 (P=0.018). Differences were found in mean epithelial thickness and inflammatory reaction in the lamina propria but did not reach statistical significance. CONCLUSIONS: In the scarred rabbit vocal fold, HGF injection is associated with decreased collagen density in the lamina propria, whereas reinjection after 10 days produces decreased collagen density and higher vessel density.