| Liver progenitor cells have drawn a great deal of attention both for their therapeutic potential and for their usefulness in exploring the molecular events surrounding liver development and regeneration. Despite the intensive studies on liver progenitors,how to improve the effectiveness of isolation and purification of liver progenitor cells, how to culture the progenitor cells in vitro for a long time and the differentiation mechanism in liver progenitor cells are still not clear.In the first part of this research, reversibly immortalized liver single-cloning cell lines were isolated from mouse E14.5 liver by single-cell cloning and infected by retrovirus SSR#69, which contains SV40T gene flanked by LoxP site at each side. For screening the reversibly immortalized liver single-cloning cell lines, the expression of marker genes in mouse liver tissues derived from embryonic day 12.5 to postnatal day 28 were performed using reverse transcription–polymerase chain reaction firstly, we found that the expression of early marker genes CD34 and Pou5f1 decreased gradually with the mature of liver tissue while expression of AFP increased in perinatal tissues and decreased after birth. At the same time, DLK was detected at the peak of expression in E16.5 liver tissue, and then dropped gradually. The expression of late marker genes Alb, CK18, TAT, and ApoB increased gradually with the mature of liver tissue as we expected. Single-cloning cell lines expressed high levels of early liver stem cell markers but low levels of late liver markers were considered as liver progenitor cells named HP14.5 and identified by morphology and differentiation potential. The result showed that the cell line HP14.5 has the characteristics of progenitor cell such as small size, high karyoplasms ratio, ability of proliferation and induced into hepatic-like cell. We also confirmed the exogenous gene SV40T is reversible. Western blot analyses showed that the target gene SV40T was integrated into HP14.5 and knocked out when the cell line HP14.5 was treated with recombinant adenovirus Ad-Cre. The same result came from the growth curve and differentiation status which estimated by luciferase reporter plasmid drived by ALB promoter. Compared with the control group treaed with Ad-GFP, the proliferation of HP14.5 treated with Ad-Cre was inhibited while the differentiation was induced. Finally, the HP14.5 cell line labeled with luciferase was subcutaneous injected in nude mice and followed up by Xenogen IVS 200 Imaging. The result confirmed that HP14.5 cell line has no tumorigenesis ability in vivo. Therefore, reversibly immortalized liver progenitor cell has been successfully isolated and established.In the second part, we sought to determine the role of RA in the regulation of hepatic differentiation. Retinoic acid (RA) signaling plays an important role in development. However, the role of RA signaling in tissue-specific progenitors is poorly understood. Using mouse liver tissues derived from embryonic day 12.5 to postnatal day 28, we found that RAR, RXR, and RXR expressed in all the samples. Expression of RAR and RXR was low perinatally and increased postnatally, whereas RAR was undetectable in prenatal tissues and increased postnatally. RA synthesis enzymes Raldh1 and Raldh2 were expressed in all tissues, while Raldh3 weakly expressed in prenatal samples but was readily detected postnatally. Nuclear receptor co-repressors highly expressed in all tissues, while expression of nuclear co-activators decreased in perinatal tissues and increased after birth. Expression of RA signaling components and co-regulators was readily detected in HP14.5. RA induced albumin promoter-driven reporter activity. RA treatment of HP14.5 cells led to a decreased expression of early progenitor markers and an increased expression of late hepatocyte markers. Furthermore, RA induced glycogen synthesis in HP14.5 cells, indicating that RA is able to promote terminal hepatic differentiation. Therefore, our results strongly suggest that RA signaling may play an important role in regulating hepatic differentiation.In brief, reversible immortalized liver progenitor cell has been successfully isolatied and established, and RA signaling may play an important role in the regulation of hepatic cell differentiation.
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