Construction of eukaryotic expression vector expressing hepatitis B virus HBsAg and EGFP fusion protein and establishment of stable transfected Chang Liver cell line / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct a eukaryotic expression vector for expressing hepatitis B virus (HBV) recombinant HBsAg-EGFP fusion protein and obtain a stable transfected Chang Liver cell line.</p><p><b>METHODS</b>The coding region of HBsAg gene of HBV was amplified by PCR and was digested by BamH I/EcoR I . This fragment was inserted into pEGFPN1 with T4 ligase and transformed E-coli TG1. The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into Chang Liver cell by Lipofectamine 2000 cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The stable transfected cell line expressing high level HBsAg-EGFP fusion protein was obtained.</p><p><b>RESULTS</b>The eukaryotic expression vector named pEGFPN1-HBsAg was successfully constructed and the stable transfected Chang Liver cell line expressing pEGFPN1-HBsAg fusion protein was obtained.</p><p><b>CONCLUSION</b>The stable transfected Chang Liver cell line could express pEGFPN1-HBsAg fusion protein, could be used to screen the proteins differentially expressed in HBsAg expression Chang Liver cells, which brought some new clues for studying the potential molecular mechanism of HBsAg protein.</p>

Subcellular distribution and translocation of hepatitis B virus core protein in HepG2.2.15 cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>The hepatitis B virus core protein has been found in nuclei, cytoplasm, or both of hepatocytes transfected with HBV DNA. It is still unclear whether intact core particles could pass through nuclear pores and what could be the mechanism regulating the subcellular localization of the core protein. This study on the distribution of core protein in hepatocytes and its translocation has a potential advantage to learn more about the HBV life cycle.</p><p><b>METHODS</b>Dimethyl sulphoxide (DMSO, 2%), which effects hepatic differentiation, and/or 1 micro mol/L heteroaryldihydropyrimidine Bay41-4109, which interferes with the assembly of core particles, were added into HepG2.2.15 cell culture system for 4 days. The hepatitis B virus core antigen (HBcAg) and hepatitis B virus surface antigen (HBsAg) were stained with fluorescent immunocytochemistry and then observed under a confocal microscope. HBcAg in cytoplasm and nuclei were respectively extracted and analyzed using Western blot. HBV covalently closed circular DNA (cccDNA) was detected by using selective PCR method.</p><p><b>RESULTS</b>The HBcAg was mostly expressed in the cytoplasm and weak signals of cccDNA were detected in the control HepG2.2.15 cells. After DMSO treatment, the expression of HBcAg in cytoplasm was increased about 2.5-fold; the expression of HBcAg and cccDNA in nuclei also increased. With the use of Bay41-4109, the signal of HBcAg in cytoplasm decreased 2/3, but it increased in the nuclei, and cccDNA decreased in the nuclei. When the HepG2.2.15 cells were treated both with DMSO and Bay41-4109, cord-liked distribution of HBsAg was observed in the cytoplasm. HBcAg in cytoplasm was decreased 1/2 but the HBcAg in the nuclei increased about 5-fold, whereas the cccDNA was almost negative.</p><p><b>CONCLUSION</b>In HepG2.2.15 cells, the core protein is mainly assembled as a formation of core particles in the cytoplasm and they are blocked by the nuclear membrane. Bay41-4109 interferes with the assembly of core particles and the dissociated core proteins are able to enter the nuclei. DMSO promotes the nuclear entry of core protein/core particles and facilitates the formation of cccDNA.</p>

Comparisons of the characteristics and mechanisms of HBV replication in QSG-7701 and HepG2 cell lines / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To gain some insights into the critical events relating to HBV transcriptional regulation by comparing HBV replicative characteristics in different cell lines.</p><p><b>METHODS</b>Hepatic cell lines QSG-7701 and HepG2 were transfected with plasmid PUC18-HBV 1.2 by standard calcium phosphate precipitation method, and 1.0 microg pSEAP2-control vector was included in the transfection procedures to serve as an internal control monitoring the transfection efficiency. Hepatitis B surface antigen (HBsAg) in the medium was detected by ELISA method and HBV DNA was quantitated using fluorescent quantitative PCR. The intracellular HBsAg and HBcAg were detected with immunofluorescent staining. The gene expression profiles of QSG-7701 and HepG2 were compared using oligonucleotide microarray; partial differentially expressed genes were verified with quantitative RT-PCR.</p><p><b>RESULTS</b>In the medium of the cultured HepG2 cells, HBsAg and HBV DNA could be detected 6 days after the transfection, whereas in QSG-7701 cells, the HBsAg and HBV DNA could be detected for 2 weeks. The HBV DNA in the culture medium of QSG-7701 was about 50 times more than that of the HepG2 cells which were kept in 1 x 10(7)copies/ml(-3) x 10(7)copies/ml for 0 to 10 days after the transfection. On the 4th day after the transfection, 20% to 30% of the QSG-7701 cells were positive with HBsAg and HBcAg immunofluorescent staining. The gene microarray analysis showed that most transcription factors involved with HBV life cycle in QSG-7701 and HepG2 cells had similar levels, whereas some factors involved with HBV transcriptional regulation and core particle disassembly, such as interleukin-6 (R=5.1340), retinoid X receptor, alpha (R=5.1268), hepatic leukemia factor (R=3.2538), serine protease PRRS23 (R=2.8356), hepatitis B virus x interacting protein (R=0.4939), serine protease inhibitor Kazal type 1 (R=0.0740) and matrix metalloproteinase 3 (negative in QSG-7701) were all differentially expressed by HepG2 cells.</p><p><b>CONCLUSION</b>Different than HepG2 cells, the QSG-7701 cells could support a high level and relatively stable HBV replication after HBV DNA transient transfection. The HBV core particles were probably recycled in the QSG-7701 cells. The differential gene expressions between QSG-7701 and HepG2 might explain the mechanism of the different HBV replication patterns. Hepatic cell line QSG-7701 might serve as a useful tool for HBV transcriptional regulation research.</p>

Cyclosporine A improves the nuclear entry of hepatitis B virus core protein in HepG2.2.15 cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The present aimed to observe the effect of phosphatase inhibitor cyclosporine A on the subcellular location and on expression of HBcAg in HepG2.2.15 cells.</p><p><b>METHODS</b>Thirty micrograms/ml of cyclosporine A (CSA) was added into HepG2.2.15 cell culture system and on days 2 and 4 HBcAg and HBsAg were respectively stained with fluorescent immunocytochemistry and observed under confocal microscope. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling method.</p><p><b>RESULTS</b>HBcAg was mostly expressed in cytoplasm in the control HepG2.2.15 cells. After 2 days CSA administration of the expression of HBcAg and HBsAg in cytoplasm significantly decreased and the signals of HBcAg in nucleus increased , whereas the HBcAg was still mainly expressed in nucleus in about 1/4 of the cells. Cell apoptosis was observed in about 30% of the cells.</p><p><b>CONCLUSION</b>CSA improves the nuclear entry of core protein. The increase of HBcAg in nucleus was likely to be related with it's phosphorylation and cell aging or apoptosis.</p>

A study on stem cell mobilization after partial liver transplantation in rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the role of stem cell mobilization on regeneration of partially grafted livers.</p><p><b>METHODS</b>Rats models with cross-sex 50% PLTx (partial liver transplantation) were established. The rats were divided into three groups: PLTx, WLTx (whole liver transplantation) and sham operation groups. Bone marrow and liver samples were collected on days 1, 3, 5, 7 postoperatively (each n = 6). The quantitative variations of the cells with stem cell markers in the bone marrow, including beta2m-/Thy-1.1+, CD45+/CD34+, Flt2/3+ and c-kit+ markers, were detected using flow cytometry. Sry gene positive cells in donor livers were detected by fluorescent in situ hybridization (FISH), and the expressions of CD34, c-kit and Thy-1.1 were detected by immunohistochemistry technique.</p><p><b>RESULTS</b>Compared with the WLTx and sham operation groups, beta2m-/Thy-1.1+, CD45+/CD34+ cells in bone marrows in the PLTx group increased on the first postoperative day and decreased on the following days. The CD34, c-kit and Thy-1.1 positive cells detected in portal tract areas peaked during the 3-5 postoperative days. CD34+/CD45+ positive cells could be detected. The expressions of CD34, c-kit and Thy-1.1 positive cells were rare in the WLTx and sham operation groups. Sry+ cells could be detected in portal tract areas and few Sry+/CD34+ and Sry+/Thy-1.1+cells were detected.</p><p><b>CONCLUSION</b>In the PLTx group, the stem cells in the bone marrow were mobilized and stem cells in the liver were activated.</p>