Construction of NTCP-Huh7 cell lines and their susceptibility to HBV / 医学研究生学报

ObjectiveThe transient expression of the functional receptor sodium taurocholate cotransporting polypeptide (NTCP) leads to the inefficiency and instability of the hepatitis B virus (HBV)-associated hepatocellular carcinoma cell model. The aim of this study was to construct NTCP-Huh7 cell lines by transfecting GV358-NTCP into Huh7 cells and identify their susceptibility to HBV.MethodsThe recombinant plasmid GV358-NTCP was obtained by ligation of GV358 and NTCP, and then transfected into Huh7 cells for the construction of NTCP-Huh7 cells. The NTCP-Huh7 cells (NTCP-Huh7 group) and Huh7 cells (Huh7 group) were infected with HBV and co-incubated with HBV for 18 hours, and the co-incubation was continued after change of the culture medium. At 2, 4, 6, 8, 10, and 12 days of incubation, the supernatant and cells were collected for measurement of the contents of HBsAg, HBeAg, HBcAg and HBV DNA in the supernatant and HBV cccDNA in the cells as well as for determination of HBV susceptibility of the NTCP-Huh7 recombinant cells.ResultsWestern blot showed stably expressed NTCP proteins in the NTCP-Huh7 cells. At 8 days of incubation, the levels of HBsAg and HBeAg were significantly higher in the NTCP-Huh7 group (0.866±0.040 and 0.603±0.053) than in the Huh7 group (0.237±0.063 and 0.209±0.112) (P<0.05), reaching the peak at 8 days and also remarkably higher in the former than in the latter group at 4, 6 and 10 days (P < 0.05). So were the levels of HBV DNA and HBV cccDNA in the NTCP-Huh7 than in the Huh7 group at 4, 6, 8, 10 and 12 days (P < 0.05). Immunofluorescence assay revealed the core antigen of HBV in the NTCP-Huh7 but not in Huh7 cells.ConclusionNTCP-Huh7 cells obtained by transfection of the GV358-NTCP recombinant plasmid into Huh7 cells are susceptible to HBV infection.

Mesenchymal stem cells transplantation:A new therapeu-tic strategy for liver failure / 医学研究生学报

Liver failure is a severe clinical syndrome and hitherto lack of effective treatment. Large numbers of studies have shown that mesenchymal stem cells (MSCs),especially umbilical cord MSCs,have a therapeutic effect on acute liver failure. Yet,the homing of MSCs in vivo affects the effectiveness of engraftment. The author presents an overview of the results of recent basic and clini-cal studies on the treatment of liver failure with MSCs and proposes a direction of development in this field,hoping to give some enlight-enment to the postgraduates and clinicians of hepatology.

Clinical efficacy of compound glycyrrhizin tablets in the treatment of children with nonalcoholic fatty liver disease / 中国当代儿科杂志

Department of Pediatrics, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. zhuchuanlong@jsph.org.cn.

Determination and clinical significance of tissue inhibitors of metalloproteinase-1 and -2 in serum of children with nonalcoholic fatty liver disease / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To examine serum tissue inhibitors of metalloproteinases (TIMP) -1 and -2 levels in children with nonalcoholic fatty liver disease and to investigate possible roles of the two markers.</p><p><b>METHODS</b>One hundred and five obese children were classified into 4 groups: simple obesity (n=44), simple nonalcoholic fatty liver (SNAFL, n=25), and nonalcoholic steatohepatitis (NASH, n=36). Serum TIMP-1 and -2 levels were measured using ELISA. Serum ALT and gamma-GT levels were measured with totally automatic enzymatic method.</p><p><b>RESULTS</b>Serum levels of TIMP-1 and gamma-GT increased with the disease development from simple obesity to SNAFL and NASH (P<0.05). Both serum TIMP-1 and -2 levels were positively correlated with gamma-GT levels (r=0.534, P<0.01; r=0.351, P<0.05, respectively). Ninety-seven percent of children in the NASH group had serum TIMP-1 levels over 2 standard deviations of healthy controls (83.35 microg/ L) compared with 76% in the SNAFL group (P<0.05). There were no significant differences in the case proportion with TIMP-2 levels over 2 standard deviations of healthy controls between the NASH and the SNAFL groups.</p><p><b>CONCLUSIONS</b>Both TIMP-1 and -2 may reflect the state of liver fibrosis in children with nonalcoholic fatty liver disease, and serum TIMP-1 appears to be more reliable.</p>

Construction of shRNA of Fulminant Hepatitis Related Gene mfgl2 and Investigation of Its Biological Effects in vitro / 中国病毒学

This study was designed to explore the RNA interference technique in inhibition of the expression of the mouse fibrinogen like protein 2 (mfgl2), which has been reported to be involved in the development a variety of diseases including fulminant viral hepatitis. A plasmid named p-mfgl2shRNA,complementary to the sequence of mfgl2 was constructed, while another short hairpin RNA (shRNA)which was a mutated form of the mfgl2shRNA sequences was used as a control. A plasmid named pEGFP-mfgl2 expressing the mfgl2-EGFP fusion protein was also constructed for the screening of the effect of p-mfgl2shRNA on mfgl2 expression. By cotransfection of p-mfgl2shRNA and pEGFP-mfgl2 or pcDNA3.1-mfgl2 expression construct into CHO cells or HeLa cells, the inhibition of mfgl2 expression by mfgl2shRNA was analyzed by direct observation through fluorescent microscopy, FACS, RT-PCR and immunohistochemistry staining. The experiments showed the significant inhibitory effect of p-mfgl2shRNA on mfgl2 expression at 48h post-transfection in both CHO and Hela cell lines with the inhibitory efficiency as high as 80.1%. The study demonstrated that the construct of p-mfgl2shRNA successfully interfered with the mfgl2 expression in vitro.

A Primary Study of the Subgroups of T Lymphocytes in MHV-3 Induced Chronic Viral Hepatitis / 中国病毒学

To study the contribution of T cell subsets in the pathogenesis of Murine hepatitis virus Type3 (MHV-3) induced chronic viral hepatitis in C3H/Hej mice, ninety C3H/Hej mice were chosen to individually receive 10 plaque forming units (PFU) of MHV-3 intraperitoneally. The changes of virus titer and pathology in liver tissue were examined by standard plaque assay and by the hematoxylin/eosin (HE) staining method from 2 days post MHV-3 infection. The ratios of T cell subsets including CD3+CD4+CD8-, CD3+CD4-CD8+, CD3+CD4-CD8-, CD3+CD4+CD25+, CD3+CD4+CD25- and CD3+CD4-CD25+ T lymphocyte of total T lymphocytes in blood, spleen and liver were examined at 0, 2, 4, 6,8, 10, 12, 15, 20, 25, 30, 40 days post MHV-3 infection by flow cytosorting. We observed that the virus titer raised and showed persistent virus duplications and inflammatory changes in the livers of C3H/Hej mice from 2 days post MHV-3 infection. The double negative T cell (DN Treg cell) and CD4+CD25+ T cell ratios increased significantly from 2 days post MHV-3 infection in C3H/Hej mice, and CD3+CD4+CD8-, CD3+CD4-CD8+, CD3+CD4+CD25- and CD3+CD4-CD25+ T cell ratios decreased accordingly. In conclusion, the changes of virus titer and pathology in the livers of C3H/Hej mice post MHV-3 suggest their contribution to viral persistence. Further characterizations of DN Treg cells are that infection indicates that MHV-3 could induce the chronic inflammation in livers of C3H/Hej mice.The increase of the DN Treg cell and CD4+CD25+ T cell ratios in C3H/Hej mice post MHV-3 infection suggests that DN Treg cells and CD4+CD25+ T cells may both have important suppressive immunomodulation functions in the development of chronic viral hepatitis and have important roles in the virus persistent infection. Further characterizations of DNT cell and CD4+CD25+ T cell are under investigation.

Construction of the p-mfgl2shRNA and its effect on mfgl2 expression in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct the siRNA plasmid for mfgl2 gene, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/zero transplantation and fetal loss syndrome, and to investigate its inhibitory effects on mfgl2 expression in vitro.</p><p><b>METHODS</b>A plasmid p-mfgl2shRNA complimentary to the sequence responsible for the functional domain of mouse fgl2 (mfgl2) was constructed. The pcDNA3.1 mfgl2 expression construct was able to show a satisfactory fgl2 protein expression. The plasmid expression pEGFP and a construct expressing irrelevant shRNA with a random combination of the p-mfgl2shRNA sequence were used as controls. A pEGFP-mfgl2 expressing mfgl2-EGFP fusion protein was also constructed for screening of the effect of p-mfgl2shRNA on the mfgl2 expression.</p><p><b>RESULTS</b>Cotransfection of p-mfgl2shRNA with pEGFP-mfgl2 decreased green fluorescent cells and the lightness of fluorescence within the cells at the 24 h, 48 h and 72 h post-transfection when compared with that in the control groups which were solely transfected with pEGFP-mfgl2. Furthermore the mfgl2 expression was significantly reduced when the pcDNA3.1 mfgl2 expression construct was cotransfected with p-mfgl2shRNA both at mRNA level by RT-PCR and protein level by RT-PCR, immunohistochemistry staining and FACS in both CHO cell and Hela cell lines.</p><p><b>CONCLUSIONS</b>The study demonstrated that the construct of p-mfgl2shRNA successfully interfered in the mfgl2 expression in vitro. It provides a basis for a further investigation of effect in vivo.</p>