Objective To assess the antibody-dependent enhancement of infection（ADE） when the cultured trophoblastic cells were infected by hepatitis C virus（HCV）. Methods We incubated the cytotrophoblasts with HCV by four different methods. Under the immunoelectron microscopy,we observed the HCV viral particles. RT-PCR method and immunohistochemistry were employed to assay the expressions of HCV NS5 and NS3 and HCV-C antigen and HCV RNA in cells or supernatant. Results The HCV viral particles were observed in the cultured cytotrophoblasts. The HCV RNA and reverse RNA and HCV NS5 and NS3 and HCV-C antigen were only intermittently detected in the culture medium or the cells incubated with HCV RNA positive serum. Conclusion The HCV viral particles can replicate in the cultured cytotrophoblasts. The ADE plays an important role when HCV viral particles enter the cultured cytotrophoblasts. Anti-HCV and/or complement probably enhanced the infection of viral particles.

Objective To investigate the inhibitory immune mechanisms of cytidine-phosphate-guanosine oligodeoxynucleotides（CpG） on liver cancer proliferation via Toll-like receptors-9（TLR9）. Methods The recombinant expression plasmid of hepatitis B virus X （HBx） were transfected into HepG2 cells and the blank plasmid-transfected HepG2 cells was the control group. 24h later,the HBx protein and the β-actin control protein were identified in the HepG2 cells by Western blotting. Then,CpG and Kupffer cells（KC）,CpG-control and KC were added into the transfected HepG2 cell culture system,respectively. 12h later,the supernatant were collected to detect interferon （IFN）-α and IFN-γ by ELISA,and the adherent HepG2 cells were collected to detect luciferase by reporter gene assay system. Results The Western blotting results showed that the HBx plasmid-transfected HepG2 cells expressed the specific HBx protein; and the dual luciferase reporter gene assay showed that the activity of HBx-mediated signaling decreased by 35% when adding KC cells and CpG into the HBx plasmid-transfected HepG2 cell culture system;and CpG activated KC cells to produce IFN-α and IFN-γ , which were increased by 8.7 folds and by 6.5 folds respectively compared with the control group. Conclusion CpG-activated KC cells could inhibit HBx-mediated signaling through the production of IFN-α and IFN-γ. It might provide a theoretical basis for HCC therapy by blocking the HBx-mediated signal pathway.

Objective To explore the relationship between CD4⁺CD25⁺ regulatory T cells and different clinical outcomes after HBV infection. Methods Fresh isolated peripheral blood mononuclear cells（PBMCs） in 26 patients with chronic hepatitis B（CHB）,15 with asymptomatic HBsAg carrier（ASC）,11 with liver cirrhosis（LC）and in 16 healthy donors were analyzed for the proportion of CD4⁺CD25⁺ regulatory T cells by using flow cytometry. Results The proportions of CD4⁺CD25⁺ regulatory T cells to CD4⁺ T cells in patients with CHB（4.40±2.76%） and ASC（4.43±2.10%） were higher than those in healthy donors（2.70±0.97%,P<0.01）;the percentage of CD4⁺CD25⁺ regulatory T cells was not correlated with serum HBV DNA level （r=0.018,P>0.05） or serum HBeAg positive or negative states （P>0.05）. Conclusion The level of CD4⁺CD25⁺ regulatory T cells in CHB patients was increased. It may be related to the chronicity of HBV infection.

Objective To sieve a assay of higher specificity and sensitivity on the spontaneous YMDD mutation in hepatitis B virus. Method Plasmids of YMDD,YVDD,YIDD were constructed with molecular methods. The sensitivity and specificity of the three assays （direct sequencing of PCR products,single probe-specific fluorescent PCR and primer-specific fluorescent PCR）were compared by detecting coexisting strains. Result Plasmids of YMDD,YVDD,YIDD which were constructed successfully were identified by restriction enzyme methods and direct sequence analysis. In mixed plasmids,a certain plasmid can be detected by direct sequencing if it covers more than 20% of the total plasmids. YMDD mutant strains only can be tested by single probe-specific fluorescent PCR when the proportion of the total strains is 50/51. The method of primer-specific fluorescent PCR has higher specificity and sensitivity when mutant strains account for more than 1/11 of the total strains. Conclusions This method of primer-specific real-time PCR is fit for the detection of the spontaneous HBV YMDD mutation.

Objective To study hepatitis B virus （HBV） genotypes and subtypes distribution in chronic hepatitis B patients. Methods Genotype/subtype-specific primer PCR was used to detect HBV genotypes and subtypes in 660 chronic hepatitis B patients from Beijing（n=474）,Shijiazhuang（n=40）,Changchun（n=55）,Dalian（n=20）,Zhengzhou（n=33）, Xi’an（n=26） and Hefei（n=12）. Results Among 660 patients with chronic hepatitis B （all with serum HBV DNA positive）,the prevalences of genotype B,C,and B/C subtype were 16.67%（110/660）,74.54%（492/660）,and 8.79%（58/660）,respectively;In genotype C-infected patients,6（1.22%） were subtype C1,473（96.14%） were subtype C2,and 13（2.64%） were C1/C2 coinfection;All genotype B were subtype Ba;There was no significant difference of HBeAg positivities or serum HBV DNA levels between subtype B and subtype C infected patients. Conclusion The common HBV genotypes in these seven cities are genotype B and C,and genotype C prevail,while subtypes Ba and C2 are predominant.

Objective To investigate the clinical and epidemiological characteristics of acute hepatitis B（AHB） in recent years. Methods To analyze the clinical data of 384 patients of AHB by using the software SPSS 13.0. Results This result indicated that the susceptible population of AHB was the patients with the age changing from 18 to 45. The main route of transmission remained unknown. The main nationality was Han. The morbidity of male was higher than that of female. The rate of recovery and improvement was 98.9%. The incidence of liver failure was 0.5%. Six months after onset,HBsAg in 97.1% cases and HBV DNA in 98.5% cases changed into negative. Conclusion The age of morbidity and the route of transmission have changed greatly. The rate of recovery and improvement is higher. Only few of the AHB patients converted to the chronic HB and severe hepatitis.

Objective To investigate the relationship among serum HBV DNA,ALT,ALB,PT levels and liver tissue injuries in patients with chronic hepatitis B. Methods Routine biochemical liver function tests and serum HBV DNA levels were assayed in 252 patients with chronic hepatitis B,and the liver biopsies were performed and the grading and staging of the liver were routinely obtained. Results There was not obvious correlation between the inflammatory grading and fibrotic staging;Serum ALT and PT in patients with G4 were significantly higher than in with G1;Serum HBV DNA levels was not correlated with histological activity index or firbosis. Conclusion Liver biopsy still plays an unique role in judging the pathogenetic condition of liver in patients with chronic hepatitis B.

Objective To evaluate the efficacy of lamivudine treatment in patients with chronic hepatits B and analysis of predicting factor. Methods Ninety patients with chronic hepatitis B were treated with lamivudine for 48 weeks. Results 21 patients （21.3%） had complete response （CR），64 patients （71.1%） had partial response （PR） and 5 patients（5.6%） were non- response（NR） in the 90 patients at the end of the regimen；In patients with baseline serum ALT≥2×upper limit of normal（ULN） and with HBeAg-negative，the rates of CR and virological response were higher，while the serum HBV DNA load had no predicting value；In 79 patients with baseline HBeAg-positive，HBeAg negativity rate was 29.6% and HBeAg/anti-HBe seroconversion rate was 9.9%；Gender and hepatitis B infection of family members were not related to the efficacy. Conclusion Lamivudine is effective in treatment of patients with chronic hepatitis B and the baseline serum ALT， HBeAg status and early response at 4 weeks have a predicting value for 48 week efficacy.

Objective To analyze the relationship between the concentration of serum sodium and prognosis of patients with chronic severe hepatitis B,to compare the difference of predictive value between serum sodium,MELD-Na score and prothrombin time（PT）. Methods 85 patients with chronic severe hepatitis B were divided into the survival group and death group. The level of serum sodium,PT,MELD-Na score were respectively recorded after admission,and analyze the correlation between them. Receiver operating characteristic curve（ROC） and area under the curve （AUC） were used to evaluate the predictive power of serum sodium,MELD-Na score and PT. Results The level of serum sodium,MELD-Na score and PT value in survival group were 136.08± 5.66mmol/L,20.42± 7.78 and 22.28± 6.37 seconds respectively,the level of serum sodium,MELD-Na score and PT value in death group were 131.15± 6.97mmol/L,34.40± 10.72 and 34.48± 10.09s respectively. The level of serum sodium of survival patients were significantly higher than the level of death patients,P< 0.001. The level of serum sodium was negatively correlated with MELD-Na scores（r=-0.673,P< 0.001） and PT （r=-0.238,P< 0.05）. The AUC for serum sodium level,MELD-Na score and the PT value are respectively 0.261,0.878 and 0.870. Conclusion Hyponatremia is an important predict factor for the prognosis of patients with chronic severe hepatitis B. Both MELD-Na scores and PT value can predict short-term prognosis of patients with chronic severe hepatitis B,and the difference between them was not significant.

Objective To observe the clinical efficacy of magnesium isoglycyrrhizinate in the treatment of patients with chronic hepatitis B. Methods The 81 patients with chronic hepatitis B were treated with 150mg magnesium isoglycyrrhizinate and 65 were treated with 80ml compound isoglycyrrhizinate for 4 weeks. Symptoms,signs and hepatic function tests were observed. Results The level of ALT dropped more rapidly in magnesium isoglycyrrhizinate-treated patients than that in control group; There was no obvious side effects in magnesium isoglycyrrhizinate group,while there was 2 cases with side effects in control group. Conclusions Clinic symptom and liver function tests improve after treatment with magnesium isoglycyrrhizinate in patients with chronic hepatitis B.

Objective To observe the therapeutic effects of embryonic bovine liver extract compound on hepatic fibrosis in patients with chronic hepatitis B. Methods 153 patients with CHB were divided randomly into three groups receiving embryonic bovine liver extract compound,BieJiaRuanGan and conventional therapy,respectively,for three months. Results The indexes of liver function and of hepatic fibrosis improved or decreased after treatment of the rwo regimen. There was no side effects in the two treatments. Conclusion Embryonic bovine liver extract compound can decrease effectively the indexes of hepatic fibrosis and improve the liver function in patients with CHB.

Objective To evaluate the clinical effects of peginterferon and ribavirin treatment on chronic hepatitis C patients with normal serum alanine aminotransferase（ALT）. Methods 20 chronic hepatitis C patients with normal ALT and 25 patients with increased ALT were treated with peginterferon（ Pegasys） and ribavirin. Curative effect were evaluated at the 12,24 and 48 weeks during the treatment. The adverse effects of the treatment were evaluated at the same time. Result All patients with normal ALT completed the whole course of treatment. Following up studies were conducted at the 24th week after the treatment. The SVR was 45%（9/20）. At the 12th week during the treatment,the EVR was 45%（9/20）. At the end of treatment,the ETVR was 55%（11/20）. Two patients had an increase in ALT and HCV RNA level after the 24 weeks of treatment. The recurrence rate was 18.2%（2/11）. All the indexes had no statistically significant difference between the normal ALT and increased ALT group. Conclusion Combination of peginterferon and ribavirin may be used for the treatment of chronic hepatitis C patients with normal serum alanine aminotransferase. The side effect of treatment is slight.

Objective To study the pathogenesis of hepatic injury induced by endotoxin （ET） in rats with nonalcoholic steatohepatitis（NASH） and the effects of traditional Chinese medicine. Methods NASH rats were induced by high fat diet and treated by low,medium and high dose of traditional Chinese medicine for smoothing liver,removing stagnation,dredging collaterals,and descending turbid substance four weeks later. These rats were sacrificed at the end of 12 weeks. Serum lipoid,ALT,ET,TNF-α and IL-1β were measured. The expressions of CD₁₄ and NF-κB in liver were observed by immunohistochemistry. The expression of LBP and TLR-4 mRNA in liver were detected by RT-PCR. ResultsAfter 12 wk,Serum ET levels in model group rats increased significantly compared with those in control group. The levels of ET,TNF-α and IL-1β in medium-dose group were lower than those of rats in control group,the differences were significant. The quantity of CD₁₄ positive staining cells in the liver of model group increased obviously compared with those of control group,and all of them were situated in the sinusoidal lining,meanwhile they were also located around central veins of hepatic lobules. CD₁₄ positive staining cells in the liver of medium-dose group were less than those of model group evidently. On high fatty diet,a few positive staining cells with NF-кB were situated diffusedly in the portal area of liver of model group. The mRNA levels of LBP and TLR-4 in the liver of model group were higher than those of control group,but they decreased in medium-dose group comparison with those of model group,the difference was significant. Conclusion Traditional Chinese medicine therapy is an effective treatment for NASH. Its mechanism of action maybe related to decreasing the level of serum ET,and down regulating endotoxin receptors of the liver and relieving inflammatory hepatic injury.

Objective To analyse causes,clinical features,diagnosis and prognosis of drug-induced liver damage in recent years in order to improve clinical doctors’ understanding of this problem. Methods we scored inpatients with drug-induced liver damage from April 2003 to April 2009 in our hospital according to Maria“clinical diagnosis scales”,and causes of 132 patients among whole patients who scored greater than or equal to 10 were retrospectively analyzed and clinically classified. Result There were more than fifty kinds of drugs from fifteen groups that could induce liver damage. Among these drugs 72.7 percent were Chinese traditional medicine,antitubercular drug,antineoplastic drug and Antibiotics,which were the major medicine that caused liver damage. Incubation period of 5 day to 356 day,the percentage of liver damage happened in fourth week,eighth week,and twelfth week was 61.4 percent,84.9 percent and 91.7 percent respectively.78.8 percent（of the total cases） were hepatocellular injury type,13.6 percent cholestatic type,and 7.6 percent mixed type. There were 27 patients（20.5%） with no clinical symptom and objective sign but abnormal Lab findings. There were 105 patients（79.5%） with clinical symptom such as fatigue（64.4%）,anorexia（53.0%）,and colored urine（41.7%）. Among 104 patients with liver damage,all had their ALT cut down more than 50 percent in 30 days and recovered their liver function in 2 months after discontinuation. Conclusion The main causes of drug-induced liver damage were Chinese traditional medicine,antitubercular drug,antineoplastic drug and Antibiotics. Mostly drug-induced liver damage occurred within 12 weeks,and he longer the drugs are used,the higher the probability of the liver damage occurs.The main damage type were hepatic cellullar damage,and its clinical feature were similar to each acute or chronic liver disease,without specificity,its prognosis were good.

introducing the idea and procedure of manufacturing“Lingjia Capsules”,and approaching professor Lingtai Wang’s opinion of the pathology of hepatic fibrosis to show his typical view and successful research experience.