Epatic stellate cell line (LX2) was conferred by Prof. Cheng (Insititute

Epatic stellate cell line (LX2) was conferred by Prof. Cheng (Insititute of Infectious Disease, Capital Medical University). LX2 cells line is a widely used hepatic stellate cell in the fibrosis investigation [17]. HepG2 and LX2 cells were cultured at 37uC in a humidified atmosphere containing 5 CO2 in Eagle’s minimum essential medium supplemented with10 fetal bovine serum. The ultimate concentration of GP73 recombinant protein added in supernatant was 1.0, 10.0, 20.0, 50.0, and 100.0 ng/ml respectively. After 48 hours coculturing, cell proliferation was evaluated with OD value, which was detected by CCK8 assay kit (Dojindo, Kumamoto, Japan), based on manufacture’s protocol.Western blotWestern blot was performed with standard protocol. Briefly, after cells cocultured with GP73 recombinant protein 48 hours, whole-cell extracts were prepared in assay buffer containing a protease inhibitor cocktail. Protein assays were performed using a BCA Protein assay kit (Pierce/Thermo Scientific, USA) according to the manufacturer’s instructions. Total protein was electrophoresed in SDS AGE gels, and transferred to nitrocellulose membranes and then blocked with 5 milk in PBS, pH 7.4 with 0.05 Tween-20, incubated with collagen I or collagen III polyclonal antibody (Santa Cruz, USA) and antirabbit secondary antibody conjugated to horseradish peroxidase (Santa Cruz., USA). GP73 was detected by 26001275 chemiluminescence.Biochemical analysisThe liver buy 57773-63-4 function tests including serum albumin, total bilirubin (TB), and alanine aminotransferase (ALT) were measured using a Roche Hitachi 717 chemistry analyzer at the central laboratory of Beijing Ditan hospital. Quantitative 842-07-9 custom synthesis determination of GP73 in serum was performed using commercially available enzyme-linked immunosorbent assay (ELISA)GP73, a Marker for Evaluating HBV ProgressionFigure 1. Serum GP73 concentration was correlated with liver stiffness (761 patients). A: Different GP73 levels were observed in patients with different groups of liver stiffness. B: serum GP73 concentration was correlated with liver stiffness. C and D: the ROC analysis of GP73 was performed on diagnosis of significant fibrosis and liver cirrhosis. The numbers after symbols “,”or “ = ” are p value. doi:10.1371/journal.pone.0053862.gStatistical analysisStatistical analysis was performed using GraphPad Prism 5.0. Student t test was used to compare the difference of serum GP73 concentrations between different patients groups (mild and significant fibrosis group). Correlation between serum GP73 concentration and liver stiffness scores were calculated using Pearson’s correlation coefficient (r). Data were expressed as mean 6 SEM. P-values ,0.05 were considered to be statistically significant. With liver stiffness value (FibroScan) or biopsy as the “gold standard”, the diagnostic performance of GP73 was evaluated by performing the Area under the ROC curve (AUROC) with 95 confidence interval (CI). For adjusting other confounders (Sex, Age, ALT, Total Bilirubin, Albumin, Platelet), we performed multivariate ordinal logistic regression analysis by SPSS 16.0.Results Patient’s characteristicsFrom Aug. 2010 to Mar 2012, 761 patients received liver stiffness measurements; 633 patients received liver biopsy, in which 472 patients with nearly normal ALT. Those patients consecutively admitted into Beijing Ditan Hospital, Capital Medical University and 302 Military Hospital. The demological materials of two populations were showed in table 1.Serum G.Epatic stellate cell line (LX2) was conferred by Prof. Cheng (Insititute of Infectious Disease, Capital Medical University). LX2 cells line is a widely used hepatic stellate cell in the fibrosis investigation [17]. HepG2 and LX2 cells were cultured at 37uC in a humidified atmosphere containing 5 CO2 in Eagle’s minimum essential medium supplemented with10 fetal bovine serum. The ultimate concentration of GP73 recombinant protein added in supernatant was 1.0, 10.0, 20.0, 50.0, and 100.0 ng/ml respectively. After 48 hours coculturing, cell proliferation was evaluated with OD value, which was detected by CCK8 assay kit (Dojindo, Kumamoto, Japan), based on manufacture’s protocol.Western blotWestern blot was performed with standard protocol. Briefly, after cells cocultured with GP73 recombinant protein 48 hours, whole-cell extracts were prepared in assay buffer containing a protease inhibitor cocktail. Protein assays were performed using a BCA Protein assay kit (Pierce/Thermo Scientific, USA) according to the manufacturer’s instructions. Total protein was electrophoresed in SDS AGE gels, and transferred to nitrocellulose membranes and then blocked with 5 milk in PBS, pH 7.4 with 0.05 Tween-20, incubated with collagen I or collagen III polyclonal antibody (Santa Cruz, USA) and antirabbit secondary antibody conjugated to horseradish peroxidase (Santa Cruz., USA). GP73 was detected by 26001275 chemiluminescence.Biochemical analysisThe liver function tests including serum albumin, total bilirubin (TB), and alanine aminotransferase (ALT) were measured using a Roche Hitachi 717 chemistry analyzer at the central laboratory of Beijing Ditan hospital. Quantitative determination of GP73 in serum was performed using commercially available enzyme-linked immunosorbent assay (ELISA)GP73, a Marker for Evaluating HBV ProgressionFigure 1. Serum GP73 concentration was correlated with liver stiffness (761 patients). A: Different GP73 levels were observed in patients with different groups of liver stiffness. B: serum GP73 concentration was correlated with liver stiffness. C and D: the ROC analysis of GP73 was performed on diagnosis of significant fibrosis and liver cirrhosis. The numbers after symbols “,”or “ = ” are p value. doi:10.1371/journal.pone.0053862.gStatistical analysisStatistical analysis was performed using GraphPad Prism 5.0. Student t test was used to compare the difference of serum GP73 concentrations between different patients groups (mild and significant fibrosis group). Correlation between serum GP73 concentration and liver stiffness scores were calculated using Pearson’s correlation coefficient (r). Data were expressed as mean 6 SEM. P-values ,0.05 were considered to be statistically significant. With liver stiffness value (FibroScan) or biopsy as the “gold standard”, the diagnostic performance of GP73 was evaluated by performing the Area under the ROC curve (AUROC) with 95 confidence interval (CI). For adjusting other confounders (Sex, Age, ALT, Total Bilirubin, Albumin, Platelet), we performed multivariate ordinal logistic regression analysis by SPSS 16.0.Results Patient’s characteristicsFrom Aug. 2010 to Mar 2012, 761 patients received liver stiffness measurements; 633 patients received liver biopsy, in which 472 patients with nearly normal ALT. Those patients consecutively admitted into Beijing Ditan Hospital, Capital Medical University and 302 Military Hospital. The demological materials of two populations were showed in table 1.Serum G.