Objective To investigate the effect of hepatitis B virus (HBV) on the immune function of natural killer (NK) cells.Methods Healthy human peripheral blood-derived NK cells were cultured alone,or co-cultured with plasmacytoid dendritic cell (pDC) (NK∶ pDC=5∶1) for 48 h with or without HepG 2.2.15-derived HBV.Cell activation was assessed by flow cytometry.The specific enzyme linked immunosorbent assay (ELISA) and intracellular cytokine staining (ICS) were used to determine the cytokine production,the cytotoxic effect of NK cells on the carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled K562 target cells as well as the granzyme and perforin levels in NK cells.Paired results were analysed using Wilcoxon signed rank test.Results HBV did not affect interleukin (IL)-12/IL-18 and IL-18/interferon α (IFNα)-induced interferon γ (IFNγ) production by NK cells when NK cells were cultured alone (both P＞0.05).However,HBV significantly inhibited pDC-induced IFNγ production by NK cells (146.1 pg/mL),while CpG enhanced pDC-induced IFNγ production by NK cells significantly (1135.4 pg/mL,P=0.0005).HBV did not affect pDC-induced NK cell activation and cytotoxicity to K562 target cells as well as intracellular granzyme and perforin levels.ConclusionHBV does not activate but inhibits pDC-induced NK cell function,which may contribute to the persistence of HBV infection.

Objective To study the expression profile of microRNA (miRNA) and the roles in pathogenesis of acute liver failure in mouse model. Methods Eighty-five BALB/c mice were divided into four groups: 40 in model group of acute liver failure were intraperitoneally injected with Dgalactosamine (D-GalN) and lipopolysaccharides (LPS); 20 in D-GalN group were injected with DGalN only; 20 in LPS group were injected with LPS only; 5 in control group were injected with saline.Liver histology of mouse was observed at hour 0, 5, 7 of injection, and sera and liver tissues were collected at hour 0, 1, 3, 5, 7, 9 of injection. Meanwhile, levels of inflammatory factors [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] in serum and liver tissue were detected by realtime polymerase chain reaction (PCR). Lock nucleic acid (LNA)-based miRNA microarray technology was used to detect the expression profile of hepatic miRNA, and the expression of miRNA was verified by real time quantification-polymerase chain reaction (RT-PCR). Mouse macrophage Raw264.7 cells were induced by LPS in vitro and the expressions of miRNA at different time points were detected.The comparison of means among groups was analyzed using one way ANOVA and the correlation were analyzed by Pearson and Spearman correlation. Results Microarray analysis found that the expression profile of miRNA during the acute liver failure changed dramatically. There were 97 miRNA in model group changed significantly compared with control group (P＜0.01), including 21 up-regulated and 27down-regulated at hour 5 and 7 of injection. Furthermore, the expressions of miR 146a and miR-155were verified by RT-PCR and found they both increased progressively over time after injection.Correlation analysis showed that miR-155 was well correlated with both TNF-α and IL-6 expressions.It was further found that miR-146a and miR-155 were both up-regulated in activated Raw264.7 cells in vitro. Conclusions The expression profile of miRNA changes during acute liver failure in mouse model. Inflammation associated-miR-146a and miR-155 are both up-regulated significantly, which indicatcs that they may play an important regulatory role in pathogenesis of acute liver failurc.

Objective To investigate whether a novel long-acting tumor necrotic factor (TNF) antagonist (soluble TNF receptor:IgG Fc [sTNFR:IgG-Fc]) can protect hepatocyte damage against liver failure caused by drugs in immunity-induced cirrhotic rats.Methods Wistar rats were repeatedly sensitized by human serum albumin (HSA) emulsified in complete freud adjuvant.The blood was collected at day 10 after the final sensitization.If anti-albumin antibody was positive,the rats were intravenously injected with HSA twice a week.After six weeks,liver cirrhosis was induced by immunity.All the model rats were divided into three groups with 15 each.Liver failure was induced with D-galactosamine/ lipopolysaccharide (LPS) intraperitoneal injection in the rats with liver cirrhosis in model group.The rats in pretreatment group were intraperitoneally injected with long-acting soluble TNF receptor p55 18 h before D-galactosamine/LPS injection.The control group were injected with 0.9％ sodium chloride.General condition,survival rate,liver function and pathological changes were all examined.Serum levels of interleukin (IL)-6,IL-22 and intrahepatic level of IL-6 were detected by enzyme linked immunosorbent assay (ELISA).The activity of Caspase 3 in hepatocyte lysis solution was measured by spectrophotography.Real-time polymerase chain reaction (PCR) was used to detect mRNA expressions of proliferating cell nuclear antigen (PCNA),bcl-2,bax and IL-22 receptor.Data were analyzed by variance analysis among groups.Results Rats in model group were dispirited with poor response after 12 hours and only 3 survived,compared with soluble TNF receptor p55 pre-treated group rats,in which all survived (P=0.029 8) with flexible response.Serum alanine aminotransferase levels in these two groups were (6 533± 360) and (105 ± 7) U/L,respectively.Hepatic regenerative nodule developed massive or submassive necrosis with septal fibrosis in model group,whereas soluble TNF receptor p55 alleviated the inflammatory and necrosis reaction of hepatic tissue.Serum IL-6 levels in model group and pretreatment group were (842.0±12.9) and (91.9±1.6) pg/mL,respectively (F=380.30,P＜0.01).Intrahepatic levels of IL-6 in these two groups were (26.2±1.2) and (11.1±0.8) pg/mL,respectively (F=176.90,P＜0.01),and serum IL-22 levels were (167.0±27.8) and (988.0±109.6) pg/mL,respectively (F=37.91,P＜0.01).Hepatic Caspase-3 activity was reduced by almost 60％ by soluble TNF receptor p55 pretreatment (F=303.70,P＜0.01) and bax expression reduced by 22％ (F=108.80,P＜0.01),while bcl-2 and PCNA expressions were up-regulated by 3.6-folds and 23.0-folds,respectively (F=115.60,P＜0.01; F=594.20,P＜0.01).Conclusions Long acting soluble TNF receptor p55 could improve survival rate,liver function and reduce inflammatory reaction of rats with liver failure induced by drugs on the basis of liver cirrhosis caused by immunity,which indicates that this drug may process a potential therapeutic value.

ObjectiveTo investigate the histological features as well as the factors influencing liver disease progression in Chinese patients with chronic hepatitis C (CHC). MethodsA total of 102 CHC patients who underwent percutaneous liver biopsy between August 2007 and May 2010 were recruited. Age, gender, body mass index (BMI) and transmission route of recruited patients were recorded. Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), HCV genotypes, HCV viral load and liver histological changes were detected. Statistical analysis was done by t test and Logistic regression. ResultsThe serum levels of ALT and AST in CHC patients with histological activity index (HAI) ≥4 were much higher, while platelet (PLT) counts were lower than those with HAI ＜4(t=2.209, 2. 298 and 2. 565, respectively; all P＜0.05). Likewise, in patients with F≥3, the serum levels of ALT and AST as well as the mean age and the duration of infection were significantly elevated compared with F ＜ 3 group ( t = 3.497, 2. 758, 2. 340 and 2. 570,respectively; all P＜0. 05), while PLT counts were much lower (t = 2. 761, P=0. 007). The unvariate predictors for HAI≥4 were female, ALT＞1 × upper limits of normal (ULN), AST level,F≥3, HCV RNA≥6 lgIU/mL and PLT counts. By mutivariate analysis, the Ishak stage score was the only independent predictor for HAI≥4 (OR 3.098, 95％CI 1.884-5. 092; P＜0.01). Finally,the univariate predictors for F≥3 were age, BMI≥24 kg/m2 , ALT＞1 × ULN, AST level, HAI≥4,PLT counts and duration of infection≥ 15 years. Multivariate analysis revealed that age (OR 1. 074,95％CI 1.006-1. 146; P=0.033), ALT level (OR 1. 035, 95％CI 1.015－1.055; P＜0.01), ASTlevel (OR 0. 969, 95％CI 0. 948－0. 990; P=0. 005), the duration of infection ≥15 years (OR 37. 215, 95％CI 5. 816－238. 127; P＜0.01) and HAI≥4 (OR 1. 939, 95％CI 1. 426－2. 636; P＜0.01) were independent predictors for F≥ 3. ConclusionAge, ALT level, AST level, duration of infection≥15 years, HAI≥4 are independent predictors for liver fibrosis.

Objective To observe the regulatory role of microRNA-1187(miR-1187)in hepatocyte apoptosis through miR-1187 targeting regulation of caspase-8 mRNA expression.Methods The acute liver failure model was established by injection of D-galactosamine plus lipopolysaccharides(LPS)in BALB/c mice.The liver tissues were collected for LNA-miRNA array analysis and functional analysis of genes targeted by miRNA.Embryonic murine hepatocyte cell line 2(BNL-CL2)was cultivated in vitro and treated with tumor necrosis factor(TNF)-α and D-galactosamine to induce the transfection of miR-1187 in transfected group or untransfected group.The expressions of miR-1187 and caspase-8 mRNA were detected by real-time polymeramse chain reaction(PCR)and caspase-8 protein was determined by Western blot.The apoptosis rate was detected by flow cytometry.The comparison of means between groups was done by t test.Results The miR-1187 signal was deceased with the development of acute liver failure.The 3'UTR of caspase-8 mRNA had direct binding sites with miR1187.In BNL-CL2 cell experiments,miR-1187 was down-regulated in untransfected group and decreased more slowly in transfected group(t=6.371,P<0.01).The expression of caspase-8 mRNA was up-regulated in untransfected group and increased less in transfected group(t=4.539,P<0.01).The apoptosis rate in transfected group was significantly lower than untransfected group(t=3.365,P<0.05).Conclusios miR-1187 is one of inhibitors of hepatocyte apoptosis.High expression of miR-1187 could regulate the expression of caspase-8 mRNA,thus inhibit the apoptosis of hepatocytes.

Objective To investigate the expression and function of retinoic acid-inducible gene Ⅰ(RIG-Ⅰ) in monocyte-derived dendritic cells (MoDC) at different stages of hepatitis B virus(HBV)infection and to explore the role of RIG-Ⅰ in the disease progression after HBV infection. Methods Peripheral blood samples were collected from 28 hepatitis B virus-infected persons, including 21 cases of chronic hepatitis B (CHB) and 7 of acute hepatitis B (AHB). Eighteen healthy subjects were recruited as controls. Purified CD14~+ monocytes were isolated by CD14 microbeads. MoDCs were induced from CD14~+ monocytes with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 for 7 days, and then were infected with vesicular stomatitis virus (VSV) to stimulate RIG-Ⅰ expression. The mRNA expression levels of RIG-Ⅰ, interferon (IFN )-promoter stimulating factor-1 (IPS-1) and IFN-β at 16 hours and 24 hours after infection with VSV were measured by real-time quantitative polymerase chain reaction (PCR). Data with normal distribution were tested by analysis of variance. Continuous variables between groups were compared using Mann-Whitney U test. Comparison among multiple groups was done by Kruskal-Wallis test. Results The expression levels of RIG-Ⅰ in MoDCs from CHB patients were significantly lower than those in AHB patients and healthy controls at 16 hours (2.44±2.03, 19. 54±3. 15, 21. 48±8. 39, respectively; F=7.451,P=0.002) and 24 hours (2. 68±2. 93, 10. 31 ±3. 88, 14. 01 ±5. 04, respectively, F = 7. 908, P = 0. 001)following VSV stimulation. The IPS-1 levels in both CHB patients and AHB patients were higher than those in healthy controls at 16 hours (2. 05±l. 08, 1. 99±1. 56, 0. 60±0. 31, respectively) F=7.246,P =0.003) and 24 hours (2. 27±2. 16, 3.24 ± 1.21, 1. 08±0. 73, respectively; F= 13. 598, P = 0. 001).Furthermore, the IFN-β expression levels were significantly lower in CHB patients compared to AHB patients and healthy controls at 16 hours and 24 hours after VSV stimulation. Conclusions The expressions of RIG-Ⅰ and IPS-1 in MoDC are abnormal in HBV infected persons, which indicates that RIG-Ⅰ signaling pathway might be blocked by HBV. The impaired function of MoDC may play a role in HBV infection and chronicity.

Objective To construct lentivirus vectors carrying 16 short hairpin RNA (shRNA) expression cassettes targeting histone acetyltransferases and provide a powerful research approach to explore the mechanism of epigenetic genes in regulating hepatitis B virus (HBV).Methods Following the rule of short shRNA primer design,eight-pair primers (A ~ H )for each gene,which had stable interfering efficiency,were designed.The annealed primers were connected to the empty lentiviral vectors of shRNA for transformation.In order to confirm the positive clones,clones were analyzed by real-time polymerase chain reaction (RT-PCR ).Then, qualified plasmids were verified by enzyme digestion technology.Four shRNA lentivirus plasmids against the same gene were mixed to build lentivirus respectively.After the virus transfected into 293T cells for 48 and 72 hours,supernatants were collected to infect HepG2.2.15 cells.The percentage of fluorescent cells were observed and assessed by microscope 72 hours after infection.Results One hundred and twenty-eight lentiviral vectors of RNA interference (RNAi)library were constructed against 16 histone acetyltransferases and more than 80% of HepG2.2.15 cells were infected with lentivirus 72 hours after infection.Conclusions Sixteen shRNA lentivirus vectors against histone acetyltransferase are successfully constructed.Thus,a solid foundation for the study of the effect of human histone deacetylase on HBV replication is established.

Objective To explore the expressions of circulating microRNAs (miRNAs) in acute liver failure mice induced by D-galactosamine (GalN)/lipopolysaccharides (LPS) and the correlation with miRNAs in the liver.Methods Forty clean grade Balb/C mice,with 32 in the model group and 8 in the control group were enrolled in the study.Liver failure was induced by intraperitoneally injection of D-GalN and LPS in mice of the model group,while mice of the control group were intraperitoneally injected with 1 mL 0.9 ％ sodium chloride solution.Serum and liver samples were collected at 0,3,5,7 hours following administration,and eight mice should be supplied to each sample,and changes of alanine aminotransferase (ALT),aspartate aminotransferase (AST) and histopathology of the liver were observed.miRNA from both the serum and the liver was extracted,miRNA expression profile in the liver at 0,5,7 hours by locked nucleic acid (LNA)-miRNA microarray was analyzed and miRNA by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was detected.Means of the two groups were compared using one-way ANOVA and correlation analyses were performed using Pearson and Spearman correlation.Results Expression of miRNAs in the liver tissue changed significantly over time with the occurrence of acute liver failure in the mice.Twenty-one miRNAs were up-regulated and 27 were down-regulated,among which miRNA-122 and miRNA-1187 were down-regulated while miRNA-146a and miRNA-155 were up-regulated.It was confirmed by the PCR assay that the expression of miRNA-122 and miRNA-1187 in the liver gradually decreased,while those in the serum were up-regulated over time.However,the expressions of inflammation associated miRNA-155 and miRNA-146a were up-regulated both in the serum and the liver after administration.The expressions of miRNA-122 and miRNA-1187 were negatively correlated between serum and liver (r=-0.477,P=0.0089,r=-0.420,P=0.231),while the expressions of miRNA-155 in serum and liver were positively correlated (r=0.678,P=0.0001).Moreover,the expressions of miRNA-122 (r=0.571,0.554) and miRNA-1187 (r=0.471,0.542) were also positively correlated with serum levels of ALT and AST (all P＜0.05).Liver and serum levels of miRNA-122 and miRNA-1187 changed significantly at 5 hours after administration,which preceded the changes of ALT/AST.Conclusions The expressions of miRNA-122 and miRNA-1187 in serum are well inversely correlated with the corresponding expressions in liver tissues during acute liver failure in mice.The changes of miRNA-122 and miRNA-1187 in the serum precede those of ALT/AST.These data suggest that serum miRNA-122 and miRNA-1187 might be the candidate serum biomarkers for early prediction of liver injury.

OBJECTIVETo investigate the changes in circulating plasmacytoid dendritic cells (pDCs) in patients with chronic hepatitis B (CHB) during the course of treatment with pegylated-interferon alfa-2s (peg-IFNa-2a) and to determine the correlations with therapeutic response.

METHODSForty-one patients with CHB who were receiving peg-IFNa-2a antiviral treatment for 48 weeks were enrolled in the study.Expression of the Toll-like receptor 9 (TLR9) on and frequency and functionality of the pDCs were analyzed at treatment weeks 0, 2, 12, 24, 36 and 48.

RESULTSAll patients exhibited an initially rapid decrease in the numbers of circulating pDCs and showed CpG-induced endogenous IFNa production within the first 2 weeks of treatment.Subsequently, all responders displayed a continuous increase in pDC numbers as well as functionality, both of which peaked around week 12 of treatment; in addition, these treatment responses were accompanied by significantly increased levels of type 1 T helper cytokines (P less than 0.05), which did not occur in the non-responders.

CONCLUSIONpDCs are involved in the initial therapeutic immune response stimulated by peg-IFNa-2a treatment.Recovery of blood pDC number and functionality may represent a predictor of favorable response to peg-IFNa-2a antiviral treatment in patients with CHB.

Objective To measure the expression of circulating microRNA (miRNA)in patients with hepatitis B virus (HBV)-related liver failure and its relationship with disease prognosis.Methods The miRNA expressions in serum of 5 patients with HBV-related liver failure and 5 healthy control subjects were compared using Exiqon miRCURY LNATM miRNA microarray.The sera from 20 patients with chronic hepatitis B (CHB),20 patients hepatitis B related cirrhosis,50 patients with HBV-related liver failure and 40 healthy persons in Ruijin Hospital were collected.The relative expression of miRNA-595 was measured using quantitative real-time polymerase chain reaction (PCR).The relative expressions of miRNAs among groups were analyzed using student t test,the correlations were analyzed by Pearson and Spearman correlation.Results Microarray informed that 92 miRNAs changed significantly in patients with HBV-related liver failure,and miRNA-595 increased most significantly.The results of real-time PCR showed that the relative expressions of miRNA-595 ,miRNA-300 and miRNA-122 were 6.03 (t=3.134, P =0.003),3.12 (t=7.221 ,P <0.01)and 2.77 (t=2.671 ,P =0.021),which were higher compared to those in healthy control group.In the analysis of the relationship between miRNA-595 expression and disease prognosis in patients with HBV-related liver failure,the relative expressions of miRNA-595 in patients with CHB,hepatitis B related cirrhosis and HBV-related liver failure were 2.26 (t =3.780,P =0.001),3.32 (t = 6.111 ,P < 0.01)and 6.03 (t = 3.134,P = 0.003),respectively,which were all increased compared to that of the healthy control.The relative expression of miRNA-595 of patients with HBV-related liver failure was 2.66 times (t=2.450,P =0.043)higher than that of patients with CHB. When dividing patients according to prothrombin activity,miRNA-595 increased significantly in patients with early stage liver failure.When dividing patients according to model of end-stage liver disease (MELD) score,MELD score was positive correlated with the expression of miRNA-595 when MELD score was under 30 (r=0.673,P =0.004).The expression of serum miRNA-595 in survival group (11 .08,n=23) was higher than that in non-survival group (3.67,n = 27,t =4.309,P =0.041).Conclusions The expressions of miRNA595 ,miRNA-300 and miRNA-122 are all increased in patients with HBV-related liver failure,especially the expression of circulating miRNA-595 at early stage of the disease.The miRNA-595 may be used as a new serum biomarker for monitoring the severity of disease.