Expression of hepatitis B virus X protein in transgenic mice.

AIM: To establish a mice model harboring hepatitis B virus x gene (adr subtype) for studying the function of hepatitis B virus X protein, a transactivator of viral and cellular promoter/enhancer elements. METHODS: Expression vector pcDNA3-HBx, containing CMV promoter and hepatitis B virus x gene open reading fragment, was constructed by recombination DNA technique. Hela cells were cultured in DMEM and transfected with pcDNA3-HBx or control pcDNA3 plasmids using FuGENE6 Transfection Reagent. Expression of pcDNA3-HBx vectors in the transfected Hela cells was confirmed by Western blotting. After restriction endonuclease digestion, the coding elements were microinjected into male pronuclei of mice zygotes. The pups were evaluated by multiplex polymerase chain reaction (PCR) at genomic DNA level. The x gene transgenic mice founders were confirmed at protein level by Western blotting, immunohistochemistry and immunogold transmission electron microscopy. RESULTS: Expression vector pcDNA3-HBx was constructed by recombination DNA technique and identified right by restriction endonuclease digestion and DNA direct sequencing. With Western blotting, hepatitis X protein was detected in Hela cells transfected with pcDNA3-HBx plasmids, suggesting pcDNA3-HBx plasmids could express in eukaryotic cells. Following microinjection of coding sequence of pcDNA3-HBx, the embryos were transferred to oviducts of pseudopregnant females. Four pups were born and survived. Two of them were verified to have the HBx gene integrated in their genomic DNA by multiplex PCR assay, and named C57-TgN(HBx)SMMU1 and C57-TgN(HBx)SMMU3 respectively. They expressed 17KD X protein in liver tissue by Western blotting assay. With the immunohistochemistry, X protein was detected mainly in hepatocytes cytoplasm of transgenic mice, which was furthermore confirmed by immunogold transmission electon microscopy. CONCLUSION: We have constructed the expression vector pcDNA3-HBx that can be used to study the function of HBx gene in eukaryotic cells in vitro. We also established HBx gene (adr subtype) transgenic mice named C57-TgN (HBx)SMMU harboring HBx gene in their genome and express X protein in hepatocytes, Which might be a valuable animal system for studying the roles of HBx gene in hepatitis B virus life cycle and development of hepatocellular carcinoma in vivo.

[Experimental study of peripheral nerve grafts for repairing of chronic spinal cord injury in adult rats].

OBJECTIVE: To explore the pathological mechanism in the repair of chronic spinal cord injury with free grafting of autoperipheral nerve tissues in rats. METHODS: The SD rats were used to establish SCI model with modified Allen method. The rats were divided into two groups at 12 weeks after the injury, each group had 20 rats. In the experimental group, the sural nerves were removed epineurium and transplanted into SCI lesion by using microsurgical technique; and in the control group, the rats were treated without any operation. The survival and differentiation of the grafts, and the ability of repairing host spinal cord were observed under the light microscope at the postoperative 4th and 12th week. Regeneration rates of nerve tracts in spinal cord were evaluated by using HRP tracing technique at the postoperative 4th and 12th week. The morphological changes were observed at section of spinal cord and the motor functions of both hind legs of rats were detected. RESULTS: In the control group, spinal cord exhibited degeneration with cicatrices and cavitates. In the experimental group, peripheral nerve was almost survived, fused with the spinal tissue and axons could regrow into or span the place of injured spinal cord. Higher number of labeled nerve tracts in spinal cord were observed in experimental group, there was significant difference when compared with the control group. Motor function of hind legs of rats recovered significantly in the treatment group. CONCLUSION: Autoperipheral nerve graft tissues transplantation could survive and integrate with the host and have repairing effects on chronic spinal cord injury in rats.

Isolation and characterization of bipotent liver progenitor cells from adult mouse.

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 from rats, equivalent progenitor cells derived from mice are relatively rare. We used retrosine treatment followed by partial hepatectomy to elicit liver progenitors in mice. From these animals showing prominent ductular reactions, mouse-derived liver progenitor cell lines (LEPCs) were isolated by single-cell cloning. Phenotypic and lineage profiling of the LEPC clones were performed using immunochemistry, reverse transcription-polymerase chain reaction, and a dual-color system comprising the reporter EGFP under the control of the cytokeratin 19 promoter and the DsRed reporter under the control of the albumin promoter. LEPCs expressed liver progenitor cell markers. LEPCs also expressed some markers shared by bone marrow-derived hematopoietic stem cells c-Kit and Thy-1 but not CD34 and CD45. When cultured as aggregates in Matrigel, LEPCs differentiated into hepatocyte upon treatment with 50 ng/ml epithelial growth factor or differentiated into biliary lineage cells upon treatment with 20 ng/ml hepatocyte growth factor. In the presence of 2% dimethyl sulfoxide and 2% Matrigel, LEPCs acquired predominantly bile lineage phenotypes, with occasional patches of cells exhibiting hepatocyte phenotypes. Upon transplantation into CCl4-injured-liver, LEPCs engrafted into liver parenchyma and differentiated into hepatocytes. Considering the amenability of the mouse to genetic manipulation, these mouse-derived LEPCs may be useful tools as in vitro models to study molecular events in liver development and regeneration and can shed light in studying the therapy potential of liver stem cells.