A recombinant hepatitis B virus (HBV) expressing NanoLuc (NL) (HBV/NL) was produced by cotransfecting a plasmid containing a 1.2‐fold HBV genome carrying the NL gene with a plasmid bearing a packaging‐defective 1.2‐fold HBV genome into a human hepatoma cell line, HepG2. We found that NL activity in HBV/NL‐infected primary hepatocytes or sodium taurocholate cotransporting polypeptide‐transduced human hepatocyte‐derived cell lines increased linearly for several days after infection and was concordant with HBV RNA levels in the cells. Treatment of the virus‐infected cells with HBV inhibitors reduced NL activity in a dose‐dependent manner. Detection of HBV/NL infection, monitored by NL activity, was highly sensitive and less expensive than detection using the conventional method to evaluate HBV infection. In addition, because we also studied host factors, this system is applicable not only for studying the HBV life cycle, but also for exploring agent(s) that regulate HBV proliferation.

Preoperative treatment with DCS combination for locally advanced gastric cancer demonstrated a sufficient R0 resection rate and a good pathological response with manageable toxicities. The DDB2/ERCC1-high phenotype, as determined by immunohistochemistry, may be useful predictor of resistance to DCS chemotherapy.

Small hepatocytes (SHs), which are known to be hepatic progenitor cells, were isolated from an adult rat liver. SHs in a colony sometimes change their shape from small to large and from flat to rising/piled-up. The aim of the present study is to clarify whether the alteration of cell shape is correlated with the maturation of SHs and whether extracellular matrix (ECM) can induce the morphological changes of SHs. We used liver-enriched transcription factors (LETFs) such as hepatocyte nuclear factor (HNF) 4 alpha, HNF6, CCAAT/enhancer binding proteins (C/EBP) alpha, and C/EBP beta, tryptophan 2,3-dioxygenase (TO), and serine dehydratase (SDH) as markers of hepatic maturation. To enrich the number of SH colonies, the colonies were isolated from dishes and replated. Replated colonies proliferated and the average number of cells per colony was about five times larger at day 9 than at day 1. When the cells were treated with laminin, type IV collagen, a mixture of laminin and type IV collagen, Matrigel or collagen gel (CG), only the cells treated with Matrigel dramatically changed their shape within several days and had reduced growth activity, whereas the cells treated with other ECM did not. HNF4 alpha, HNF6, C/EBP alpha, C/EBP beta, and TO were well expressed in the cells treated with Matrigel. Furthermore, addition of both glucagon and dexamethasone dramatically induced the expression of SDH mRNA and protein in the cells treated with Matrigel. In conclusion, morphological changes of SHs may be correlated with hepatic maturation and basement membrane (BM)-like structure may induce the morphological changes of SHs.

The morphogenesis and movement of bile canaliculi (BC) are not well understood. This is because culture of hepatocytes that maintain polarity of cell membranes and possess highly differentiated functions has never been successful. We found that small hepatocytes (SHs), which are known to be hepatic progenitor cells, could proliferate and differentiate into mature hepatocytes and that BC-like structures developed between rising/piled-up cells. We investigated how BC-like structures developed with maturation of SHs and whether the structures were functionally active as BC. Hepatic cells, including SHs, were isolated from an adult rat liver and cultured. Immunocytochemistry and immunoblotting for BC proteins, such as ectoATPase, 5'-nucleotidase, dipeptidylpeptidase IV, and multidrug-resistance associated protein 2, were examined and time-lapse microscopy was used for the observation of BC contractions. Secretion of bilirubin into the reconstructed BC was also observed. The results of immunocytochemistry, immunoblots, and immunoelectron micrographs revealed that BC proteins were localized in the intercellular space that coincided with BC-like structures reconstructed between rising/piled-up cells. Tight junction-associated protein ZO-1 was also expressed along the BC-like structures. Bilirubin added to the medium were secreted into BC-like structure and accumulated without leakage. Time-lapse microscopy showed continuous contractions of reconstructed BC. In conclusion, BC-like structures reconstructed by SHs may be functional with membrane polarity, secretory ability, and motility. These results show that this culture system may suitable for investigating the mechanism of the formation of BC and their functions.

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