Objective To explore whether the polymorphisms within the tumor necrosis factor alpha (TNF-?) and TNF-? gene are associated with the clinical types of patients with chronic hepatitis B virus (HBV) infection. Methods By using a restriction fragment length polymorphism (RFLP) assay of polymerase chain reaction (PCR) products, the single nucleotide polymorphisms (SNPs) within TNF-? and TNF-? gene among 56 patients with chronic severe hepatitis B and 71 patients with chronic mild hepatitis B or asymptomatic carriers as well as 90 healthy controls were analyzed. The TNF-? concentration of serum in 56 patients with chronic severe hepatitis B and 30 healthy controls were determined by radio immunity assay (RIA). Results The frequencies of the TNF1/2 genotype and the TNF2 allele were significantly increased in patients with chronic severe hepatitis compared with healthy controls (25% vs 11.1%,P=0.028;12.5% vs 5.6%,P=0.036) and patients with chronic mild hepatitis B and asymptomatic carriers (25% vs 8.5%,P=0.011;12.5% vs 4.2%,P=0.015). Furthermore, heterozygotes carrying TNF2 allele had significantly higher levels of serum TNF-? than homozygotes for the wild type allele among all patients with chronic severe hepatitis B (P

Objective To evaluate the in vitro inhibitive activities of mycophenolic acid against hepatitis B virus in vitro. Methods The different concentrations of the test drugs in DMEM were added to the well with the monolayer growth of HepG2.2.15 cells. The culture medium was refreshed with DMEM containing the appropriate drugs every 3 days. At 3rd, 6th and 9th days, the supernatant was collected for HBsAg quantitative assay, total RNA was isolated from cells for RT-PCR and realtime-PCR assays. Results It was found no significant anti-HBV effect of mycophenolic acid on concentration of 2～250 ?g/ml, and mini inhibitory efficacy of HBV replication on concentration of 1250 ?g/ml. There was significant anti-HBV effect of lamivudine on concentration of 5 ?g/ml. When combined with lamivudine mycophenolic acid can significantly potentiate anti-HBV activities of lamivudine, and it can get maximal inhibition effect on concentration of 10 ?g/ml. Conclusions Mycophenolic acid could inhibit HBV replication and potentiate anti-HBV activities of lamivudine.

Objective On the basis of the lasted clinical experience,our group will discuss the treatmented mechanism of Chinese herb kang-xian-er-hao(KXEH) ameliorate hepatic cirrhosis. Methods Male wistar rats were divided into five groups,excepted for normal group N,the remnant four groups were all given intraperitoneal injection of porcine serum((0.5) ml/time,2 times/week,total 12 weeks).In KXEH early treatment group B,the rats were fed with KXEH by gavage,1 g/100 g,once a day at the third week.In KXRH late treatment group C,the rats were fed with KXEH by gavage,1 g/100 g,once a day at the ninth week.In ?-interferon treated group D the rats were subcutaneous injection ?-interferon((0.1) million) every day at the ninth week.The model group A and normal group N were fed with the same amount of saline by gavage.The rats were killed at the end of the twelfth weeks,the formation of liver fibrosis was observed with HE stain and Masson stain.The expression of Smooth muscle actin(SMA) was observed by immunohistochemistry.As well as SMA,collagen Ⅰ、Ⅲ mRNA and Smad3 mRNA,which is TGF-?1 downstream signal,were detected in liver samples with RT-PCR assay. Results In KXEH treated group B and C,the body weight was heavier,the size of liver and spleen was smaller and the ratio of liver weight/body weight and spleen weight/body weight was decreased compared with the model group A(P

Objective To evaluate the in vitro inhibiting activities of interferon-alpha (INF-?) against hepatitis B virus (HBV). Methods HepG2 2 2 15 cells were treated with INF-?. At 3rd, 6th and 9th days after treatment, the supernatant was collected for HBsAg quantitative assay, and total RNA of the cells was isolated for RT-PCR and realtime-PCR assay of HBV mRNA. Results There were no significant differences in HBsAg titer and virus mRNA level at 3rd and 6th days between the experimental and control groups. At 9th day, HBsAg titer and virus mRNA level in INF-? group were significantly lower than those in control group. And 500IU/ml INF-? could get maximal inhibiting effect against HBV. Conclusion INF-? can inhibit transcription and expression of HBV gene in HepG2 2 2 15 cells, which indicated that INF-? can inhibit HBV replication directly.

Objective To establish a multiplex polymerase chain reaction (M-PCR) assay to detect HBV DNA in the peripheral blood mononuclear cells(PBMCs) of chronic HB patients. Methods One pair of primer amplifying HBV genome DNA and another pair of primer amplifying HBV covalently closed circular DNA (cccDNA ) were added to one PCR reaction to detect HBV DNA in PBMCs. Results Various forms of HBVDNA including total DNA and cccDNA could be amplified simultaneously. Among the 30 chronic HB patients, both the HBVDNA and HBVcccDNA in the PBMCs of 23 patients were detected, the positive rate of which was 76.6%. The positive rate of HBV cccDNA accounted for 82.1% of total HBV DNA positive rate. Conclusion HBVDNA in the PBMCs could partially replicate. The M-PCR was successfully set up to amplify HBV genome DNA and HBV cccDNA simultaneously.

Objective To construct the recombinant plasmid carrying human sTNFR1 cDNA, and express sTNFR1 in E. Coli JM109. Methods Total RNA was extracted from Hela cells, and used as a template to amplify human sTNFR1 cDNA by RT-PCR. The PCR products were cloned into T vector, and then sTNFR1 cDNA fragment was subcloned into a prokaryotic expression plasmid pMAL-c2x. The recombinant plasmid was transferred into E. Coli JM109, and induced by IPTG to express fusion protein sTNFR1-MBP. sTNFR1-MBP was purified by amylose resin affinity chromatography(ARAC), and analyzed by SDS-PAGE. Results A 558 bp human sTNFR1 cDNA was amplified by RT-PCR, and successfully inserted into plasmid pMAL-c2x. sTNFR1-MBP was produced in E.Coli after IPTG induction, and a 66 KD sTNFR1-MBP was purified by ARAC. [WTHZ]Conclusion Recombinant plasmid carrying human sTNFR1 cDNA was successfully constructed and epxressed in E. Coli JM109.

Objective To construct an eukaryotic expression vector of human sTNFR1 and to investigate its inhibitory effects to the bioactivity of TNF-?.Methods The total RNA was extracted from HeLa cells and used as a template to amplify human sTNFR1 gene by reverse transcription polymerase chain reaction(RT-PCR).The PCR products were cloned into T vector and sub-cloned into vector pcDNA3.1(-),an eukaryotic expression vector.The recombinant plasmid pcDNA3.1(-)-sTNFR1 was transfected into QSG7701 cells by using lipofectamine,RT-PCR was performed to detect the expression of sTNFR1,MTT was used to observe sTNFR1 gene 's inhibitory effect on TNF-?.Results QSG7701 has a higer expression level of sTNFR1 mRNA than pcDNA3.1(-) trancfected control.The cytotoxic effect of TNF-? was inhibited to the extent of 64.8% when its concentration was 100 ?g/L.Conclusion We constructed the eukaryotic expression vector containing human sTNFR1 gene and the cytotoxicity of TNF-? is inhibited in pcDNA3.1(-)-sTNFR1/QSG7701 cells.

Objective To observe the effects of truncated type Ⅱ receptor of TGF ? 1 expressing plasmid on rat liver fibrosis. Methods An eukaryotic expression plasmid of a truncated type Ⅱ receptor of TGF ? 1 was constructed by recombinant DNA technique. Sequence analysis of this recombinant plasmid revealed that it contained the complete extracellular and transmembrane domains of TGF ? 1 Ⅱ receptor but lacked most of the cytoplasmic kinase domain. The recombinant plasmid encapsulated by liposome was transferred into the rats by intraperitoneal injection and its effects on the grade of the pathologic fibrosis and the levels of HA,LN,PCⅢ,Ⅳ C were observed. Results The levels of serum HA,LN,PCⅢ,Ⅳ C were lower in the early treatment group than those in the model control group ( P 0.05). Either in the early or later treatment group, the grade of liver fibrosis was decreased apparently compared with the model control group ( P

Objective To study the complexity of S region qusispecies in various disease stages of chronic hepatitis B virus(HBV) infection and its relation to disease activity. Methods Serum samples were obtained from 112 patients with chronic hepatitis B virus infection;22 with chronic carries(ASC),30 with chronic mild or moderate hepatitis(CH),60 with fulminant hepatitis failure(FHF). HBV qusispecies populations were separated by the single strand conformation polymorphism (SSCP) method targeted the S region and DNA sequencing analysis. Results The number of SSCP bands detected in the patients with ASC、CH and FHF was 1.45?0.13,3.70?0.22 and 5.93?0.24, respectively. There was a statistically significant difference in the number of quasispecies among various disease stages ( P

In order to study the affection of neutralizing antibody on the therapeutic effect of IFN? -2b,NBA was used to detect the neutralizing antibody to against IFN in the sera from 27 patients with chronic hepatitis B.The results indicated that neutralizing antibody against IFN was present in 15 of 27 patients who had been treated with IFN?-2b,and the overall frequency of neutralizing antibody was 55.6%(15/27).At the end of IFN therapy,HBV DNA was undetectable in 4 of the 15 patients(4/15, 26.7%)with neutralizing antibody.By contrast.HBV DNA became undetectable in 10 of the 12 patients (10/12,83.3 % ) without neutralizing antibody (P