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Hepatitis B virus (HBV) is a worldwide epidemic pathogen that causes hepatitis B. On‐site screening the HBV infection is of critical importance for preventing and diagnosing HBV infection. In this paper, a simple, visual, and rapid method for on‐site detection of HBV‐DNA has been developed. This method is based on betaine‐assisted recombinase polymerase assay and followed with naked‐eye detection via lateral flow assay (BRPA‐LF). Result show that nonspecific amplification is prone to occur in recombinase polymerase amplification (RPA) if the assay was performed with serum sample without purification. This problem has been addressed by adding 0.8 M of betaine to the RPA reactions. It was demonstrated that BRPA‐LF can detect 1,000 copies of HBV‐DNA in 50 μL mixture, and achieved 90% sensitivity and 100% specificity for serum sample detection. These results demonstrated that BRPA‐LF can resist serum interference and has great potential for on‐site screening of HBV infection. [ABSTRACT FROM AUTHOR]

Aims: Voriconazole is a broad‐spectrum antifungal agent for the treatment of invasive fungal infections. There is limited information about the pharmacokinetics and appropriate dosage of voriconazole in patients with liver dysfunction. This study aimed to explore the relationship between voriconazole trough concentration (Ctrough) and toxicity, identify the factors significantly associated with voriconazole pharmacokinetic parameters and propose an optimised voriconazole dosing regimen for patients with liver dysfunction. Methods: The study prospectively enrolled 51 patients with 272 voriconazole concentrations. Receiver operating characteristic curves were used to explore the relationship between voriconazole Ctrough and toxicity. The pharmacokinetic data was analysed with nonlinear mixed‐effects method. Dosing simulations stratified by total bilirubin (TBIL, TBIL‐1: TBIL < 51 μmol/L; TBIL‐2: 51 μmol/L ≤ TBIL < 171 μmol/L; TBIL‐3: TBIL ≥ 171 μmol/L) were performed. Results: Receiver operating characteristic curve analysis revealed that voriconazole Ctrough of ≤ 5.1 mg/L were associated with significantly lower the incidence of adverse events. A 1‐compartment pharmacokinetic model with first‐order absorption and elimination was used to describe the data. Population pharmacokinetic parameters of clearance, volume of distribution and oral bioavailability were 0.88 L/h, 148.8 L and 88.4%, respectively. Voriconazole clearance was significantly associated with TBIL and platelet count. The volume of distribution increased with body weight. Patients with TBIL‐1 could be treated with a loading dose of 400 mg every 12 hours (q12h) for first day, followed by a maintenance dose of 100 mg q12h administered orally or intravenously. TBIL‐2 and TBIL‐3 patients could be treated with a loading dose of 200 mg q12h and maintenance doses of 50 mg q12h or 100 mg once daily and 50 mg once daily orally or intravenously, respectively. Conclusions: Lower doses and longer dosing intervals should be considered for patients with liver dysfunction. TBIL‐based dosing regimens provide a practical strategy for achieving voriconazole therapeutic range and therefore maximizing treatment outcomes. [ABSTRACT FROM AUTHOR]

Background: C-X-C chemokine receptor type 5 (CXCR5) + CD8 + T cells represent a unique immune subset with dual roles, functioning as cytotoxic cells in persistent viral infections while promoting B cell responses. Despite their importance, the specific role of CXCR5 + CD8 + T cells in chronic hepatitis B (CHB), particularly during interferon-alpha (IFN-α) treatment, is not fully understood. This study aims to elucidate the relationship between CXCR5 + CD8 + T cells and sustained serologic response (SR) in patients undergoing 48 weeks of pegylated IFN-α (peg-IFN-α) treatment for CHB., Aim: To elucidate the relationship between CXCR5 + CD8 + T cells and sustained SR in patients undergoing 48 weeks of peg-IFN-α treatment for CHB., Methods: This study enrolled 60 patients with hepatitis Be antigen (HBeAg)-positive CHB undergoing 48 weeks of peg-IFN-α treatment. Participants were assessed for eligibility based on criteria such as persistent HBsAg-positive status for at least six months, HBeAb-negative, hepatitis B virus DNA levels exceeding 2 × 10 4 copies/mL, and alanine aminotransferase (ALT) levels between 2 and 10 times the upper limit of normal. Blood samples were collected at baseline and at weeks 12, 24, 48, and a 24-week treatment-free follow-up (week 72) to measure serum interleukin (IL)-21 concentration via ELISA and to analyze CXCR5 and programmed death-ligand 1 (PD-L1) expression on CD8 + T cells by flow cytometry, CXCR5 is a chemokine receptor that directs immune cells to specific tissues, while PD-L1 is a protein that regulates immune responses by inhibiting T cell activity., Results: Patients with CHB exhibited significantly lower levels of circulating CXCR5 + CD8 + T cells compared to healthy controls ( P < 0.01). Notably, CXCR5+CD8+ T cells were prominently expressed in patients who achieved sustained SR compared to non-SR (NSR). A significant correlation was observed between CXCR5 and PD-L1 expression ( r = -0.189, P = 0.002). However, there was no significant correlation between serum IL-21 levels and CXCR5 + CD8 + lymphocytes ( r = -0.03, P = 0.625) or serum ALT levels ( r = 0.026, P = 0.678)., Conclusion: The enhanced expression of CXCR5 + CD8 + T cells in patients achieving HBeAg seroconversion during IFN-α treatment suggests that these cells play a crucial role in antiviral immune responses against hepatitis B. This study highlights the potential of CXCR5 + CD8 + T cells as immune regulators in CHB, which may inform future therapeutic strategies to optimize antiviral treatments., Competing Interests: Conflict-of-interest statement: The authors disclose no conflicts., (©The Author(s) 2025. Published by Baishideng Publishing Group Inc. All rights reserved.)

Objective: To investigate the dynamic changes in expression of programmed death (PD)-1, Toll-like receptor (TLR)3, and TLR4 on the surface of peripheral blood mononuclear cells (PBMCs) in patients with chronic hepatitis C (CHC) that occur in response to pegylated-interferon alpha-2a (peg-IFNalpha-2a) plus ribavirin (RBV) combination therapy, and to analyze the relation to achievement of sustained virological response (SVR). METHODS Twenty-three CHC patients and 10 healthy controls were enrolled in the study. All CHC patients underwent 48 weeks of combination therapy with peg-IFNalpha-2a (180 microg, subcutaneous injection, once weekly) plus RBV (15 microg/kg, oral, once daily). Total PBMCs were isolated from both groups (CHC patients at treatment week 0, 12, 24, and 48 and post-treatment week 24; controls at enrollment) and subjected to flow cytometric analysis of PD-1, TLR3, and TLR4 surface expression. In addition, serum levels of alanine aminotransferase (ALT) and hepatitis C virus (HCV) RNA levels were analyzed by enzymatic assay and the AmpliPrep/COBAS (Roche) nucleic acid amplification test, respectively. SVR was defined as undetectable levels of HCV RNA at post-treatment week 24. Intergroup differences were assessed by one-way ANOVA., Results: The expression ratios of PD-1, TLR4 and PD-1: TLR4 on PBMCs were significantly higher in CHC patients before therapy than in the healthy controls (45.20 +/- 7.12% vs. 16.82 +/- 4.13%, 58.45 +/- 15.13% vs. 21.09 +/- 2.89%, and 35.54 +/- 7.69% vs. 14.12 +/- 2.89%; all P < 0.05). In contrast, the expression ratios of TLR3 and PD-1:TLR3 were slightly, but not significantly, higher in CHC patients before therapy than in the healthy controls (P > 0.05). During the course of peg-IFNalpha-2a plus RBV combination therapy, the expression ratios of PD-1 and TLR4 on PBMCs showed a decreasing trend, while TLR3 expression showed an increasing trend. Furthermore, CHB patients who achieved SVR at post-treatment week 24 had a significantly different expression ratio of PD-1 and TLR3 than those who did not achieve SVR (P < 0.05)., Conclusion: Surface expression of PD-1, TLR4, and PD-1:TLR4 is up-regulated in the total PBMCs of CHC patients. Peg-IFNalpha-2a plus RBV treatment-induced suppression of HCV replication results in a significant reduction in PD-1 and TLR4 expression on the surface of PBMCs, but a remarkably elevated level of TLR3 expression. The dynamic change in PD-1 and TLR3 expression on PBMCs that occurs during antiviral therapy may be related to achievement of SVR.

Objective: To observe the expression variations and influencing factors of programmed death one (PD-1) and DNA demethylation of PD-1 promoter on peripheral blood mononuclear cells (PBMCs) in chronic hepatitis B (CHB) patients and further investigate the relationship between the demethylation pattern of PD-1 gene in promoter region and the PD-1 expression on PBMC in CHB patients., Methods: A total of 162 subjects, including 144 CHB patients and 18 healthy blood donors, were enrolled. The expression of PD-1 on PBMCs was detected by flow cytometry. And the serum HBV markers, HBV DNA load and liver function were also measured. DNA of PBMCs was treated with sodium bisulfite; the PD-1 promoter fragments were amplified by polymerase chain reaction (PCR) and then transformed into Escherichia coli. Positive clones were selected for sequencing and the methylation status of fragments of PD-1 promoter was examined., Results: With the PD-1 expression in normal controls (10.8% ± 4.4%) as a baseline level, the expression of PD-1 in CHB patients significantly increased. In CHB patients, the serum expression of PD-1 in PBMCs from patients with positive HBeAg (27.1% ± 18.4%) was much higher than that from those with negative HBeAg (19.6% ± 15.6%). And the expression level of PD-1 was not correlated with serum HBV DNA load and serum level of alanine aminotransferase. The results of bisulfite genomic sequencing showed that demethylation probability of some CG points in PD-1 promoter region (-601, -553, -538, -483, -463, -317 bp) were significantly correlated with PD-1 expression level (P < 0.05)., Conclusion: The demethylation pattern of PD-1 gene in promoter region is associated with the PD-1 expression on PBMC in CHB patients.

Objective: To investigate the effect of hepatitis C virus (HCV) strain JFH1 on expression of the human gene, growth arrest and DNA damage-inducible gene 45 alpha (GADD45a), in infected hepatoma cells., Methods: HCV JFH1 RNA-containing supernatants were used to infect the human hepatoma cell line, Huh7.5.1; infection was confirmed by Western blot detection of the HCV-encoded non-structural 5A (NS5A) protein and core protein. Infection-induced changes in GADD45a mRNA and protein expressions were measured by real time PCR using SYBR Green and Western blotting, respectively. Significance of differences between the levels detected in JFH1-infected or uninfected Huh7.5.1 cells was analyzed by single factor analysis of variance testing., Results: The HCV infection system was successfully established, as evidenced by expression of NS5A protein and core protein. The GADD45a mRNA and protein levels were significantly down-regulated in JFH1-infected Huh7.5.1 cells, by 0.57+/-0.09 and 0.28+/-0.03, respectively, as compared to levels in uninfected Huh7.5.1 cells (F values were 75.407 and 560.04, respectively; P less than 0.01)., Conclusion: HCV inhibits the mRNA transcription and protein expression of host GADD45a, which may contribute to the pathogenesis of hepatocellular carcinoma caused by HCV infection.

Unlabelled: To observe the efficacy of adefovir dipivoxil(ADV) in combination with Anluohuaxian capsule in the treatment of chronic hepatitis B (CHB) patients., Methods: 72 cases with CHB were randomly divided into two groups. 36 cases of treatment group were given ADV combined with Anluohuaxian capsule for 48 weeks. 36 cases of control group were given ADV. The levels of serum ALT, AST, Alb, TBil, HA, LN, CIV, HBV DNA and hepatic tissue were compared before and after being treated., Results: After 48 weeks treatment,the liver function, serum fibrosis index and histology of treatment group and control group all have improved. After treatment, the two groups in the levels of ALT(t=0.746, P=0.342), AST (t=0.369, P=0.713), TBil (t=0.146, P=0.684), Alb(t=0.148, P=0.883), liver tissue inflammation mobility scoring (t=1.666, P=0.100) and HBV DNA negative rate (x2=0.141, P=0.708) were no evident difference.The level of HA, LN, CIV were significantly lower in treatment group(101.58+/-30.11, 147.89+/-41.72, 38.75+/-9.50) compared with control group(182.25+/-117.59, 181.50+/-56.96, 74.92+/-31.14) (P less than 0.05). After the treatment, the liver tissue fibrosis scoring was significantly lower in treatment group (10.61+/-2.37) compared with before the treatment (12.28+/-3.16) (P less than 0.05).There was no difference found between after the treatment (11.36+/-2.93) and before the treatment (12.17+/-3.01) in control group (P more than 0.05)., Conclusions: The results show that the treatment with ADV in combination with Anluohuaxian capsule can play promoting antifibrotic effect and significant improved liver histology of chronic hepatitis B patients.

Objective: To investigate whether the nonstructural protein 5A (NS5A) encoded by the hepatitis C virus RNA genome affects the expression of hepcidin gene., Methods: HCV NS5A expression plasmid (pCN5A) and pRc/CMV were transfected into QSG7701 cells individually, RT-PCR was employed to detect the HCV NS5A and hepcidin mRNA transcription. Western blot was used for detection of HCV NS5A and hepcidin proteins. Iron was stained to evaluate the intracellular iron level., Results: HCV NS5A plasmid was successfully transfected into QSG7701 cells, which was evidenced by HCV NS5A mRNA and protein from the transfected cells. The hepcidin mRNA relative quantification in untransfected cells, pRc/CMV transfected cells and pCNS5A transfected cells were 0.711+/-0.049, 0.718+/-0.052 and 0.264+/-0.030 respectively. The transcription of hepcidin mRNA decreased remarkably in the cells transfected with pCNS5A plasmid as compared to the untransfected cells and pRc/CMV transfected cells (P less than 0.01). The level of hepcidin protein expression was found also significantly lower in the pCN5A plasmid transfected cells as compared to the untransfected cells and pRc/CMV transfected cells. The intracellular iron staining was remarkably higher in the pcNS5A transfected cells than untransfected or pRc/CMV transfected cells., Conclusions: HCV NS5A inhibits the transcription of hepcidin mRNA and expression of hepcidin protein, inducing hepatic intracellular iron storage.

Background: Chronic severe hepatitis B patients often have limited survival. This investigation aimed to evaluate the short-term effects of nucleoside analog therapy on chronic severe hepatitis B., Methods: We retrospectively, randomly collected the data of 129 chronic severe hepatitis B patients: 55 were treated with entecavir, and the remaining 74 were not treated with nucleoside analogues., Results: No significant difference in short-term survival rate was found between the group treated with entecavir and that treated without nucleoside analogues. Although entecavir greatly reduced HBV replication in different periods of therapy (P<0.001), the model for end-stage liver disease (MELD) score and liver function (alanine aminotransferase, albumin, bilirubin, prothrombin time) showed no significant change. No significant differences were found in MELD scores and liver function in patients with different HBV DNA levels (< or =10(4) copies/ml, >10(4) to <10(6) copies/ml, > or =10(6) copies/ml). Nor correlation was observed between HBV DNA levels and MELD scores in different periods of therapy (P>0.05). The HBV DNA levels of patients who survived for over 3 months or less than 3 months were not significantly different either. However, the MELD score and parameters of liver function (albumin, bilirubin, prothrombin time) were different between the two groups (P<0.05)., Conclusion: These results suggest that short-term suppression of HBV replication may not slow down the progression of liver failure in patients with chronic severe hepatitis B.

Objective: To investigate the effects of different mutated sites in the vpr gene of HIV on the apoptosis of host cells, and the possible mechanism thereof., Methods: Fourteen HIV-1 vpr fragments were obtained from HIV-infected persons. Eukaryotic expression vector pcDNA3.1 (+) plasmid was extracted, the PCR purified product was double-cut by HindIII and BamH, and the cut products were ligated to vectors, thus establishing the JM109 competent cells. Sequencing was used to confirm the reconstruction of pcDNA-vpr eukaryotic expression vectors that were then transfected into HeLa cells. Blank vectors were transfected as control group. Cells were harvested after 24 hours and underwent Hoechst 33258 staining and observed under fluorescence microscope. Annexin-FITC-PI staining and flow cytometry were used to observe the percentage of apoptosis. The caspase-3 activity was detected by enzyme labeling instrument., Results: The apoptotic rates shown by Hoechst and annexin--FITC-PI staining methods, and caspase-3 activity levels of the HeLa cells transfected with the gene fragments with mutated sites 70, 85, 86, and 94 cells were all lower than the cells transfected with the gene fragments without these mutated sites. The apoptosis causing ability levels of the No 1-7 recombinant plasmids (all of the Vpr AE subtype) were all lower than those of the No 8-14 plasmids (of Vpr B, AB, C, and C/BC subtypes)., Conclusion: The apoptosis causing ability of the HIV with the vpr sequence with mutated sites 70, 85, 86, 94 is significantly lower than those without these sites. AE subtype induces lower apoptotic behavior in the hoist cells, and decreased activation of the caspase-3 pathway may be one of the mechanisms.

Aim: To study the effect of Hepatitis C virus non-structural 5A (HCV NS5A) on IFNalpha induced signal transducer and activator of transcription-1 (STAT1) phosphorylation and nuclear translocation., Methods: Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescence was performed to observe if there was any difference in IFNalpha-induced STAT1 phosphorylation and nuclear translocation between HCV NS5A-expressed and non-HCV NS5A-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells., Results: Expression of HCV NS5A was found in the cytoplasm of P(CNS5A-transfected ) Huh7 cells, but not in the PRC/CMV transfected or non-transfected cells. STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction. STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to P( RC/CMV-transfected ) and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot., Conclusion: HCV NS5A expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-alpha is able to induce STAT1 phosphorylation and nuclear translocation, and this effect is inhibited by HCV NS5A protein, which might be another possible resistance mechanism to interferon alpha therapy.

Objective: To study the effect of HCV NS5A on the Janus kinase (JAK)/signal transducer and activation of transcription (STAT1) phosphorylation and translocation induced by IFN alpha-2b and to understand the possible molecular mechanism of HCV interferon resistance., Methods: Hepatocellular carcinoma cell line Huh7 was transiently transfected with HCV NS5A protein expression plasmid pCNS5A and blank plasmid pRC/CMV respectively; the cells of the same cell line, without transfection, served as controls. Immunocytochemistry was used to prove the successful transfection. Immunofluorescence and Western blot were performed to observe the difference in STAT1 phosphorylation and nuclear translocation between HCV NS5A-expressed and non-HCV NS5A-expressed cells after 30 minutes of IFN alpha-2b induction., Results: HCV NS5A protein was detected in the cytoplasm of Huh7 cells transfected with pCNS5A, indicating the successful transfection. In comparison to the blank plasmid-transfected and non-transfected group, STAT1 phosphorylation and sequential nuclear import were reduced in the presence of HCV NS5A protein., Conclusion: HCV NS5A can, to some extent, inhibit phosphorylation and nuclear translocation of STAT1 in IFN alpha-2b-induced JAK/STAT pathway, which may be a possible mechanism of HCV interferon resistance.

Objective: To investigate whether the nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus RNA genome could affect the expression of the survivin gene., Methods: HCV NS5A expression plasmid (pCNS5A) was transfected into HepG2 cells using a lipofectin reagent and the HCV NS5A protein was detected using immuno-histochemistry staining. Survivin mRNA was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and the expression of survivin protein was detected using Western blot., Results: HCV NS5A protein was detected in the cytoplasm of the HepG2 cells transfected with pCNS5A, and the expressions of survivin mRNA and survivin protein were both up-regulated in the presence of the HCV NS5A protein., Conclusions: HCV NS5A protein can stimulate survivin protein expression, and this might be the result of the strengthening of the survivin gene transcription.

Objective: To evaluate the clinical effect and side-effect of interferon-alpha (IFN-a) and ribavirin (RBV) combination therapy for Chinese patients with co-infection of hepatitis C virus (HCV) and human immunodeficiency virus (HIV), and to compare them with only HIV infection patients., Methods: 10 patients with HCV-HIV and 17 patients with only HCV infection received 5 million units of IFNalpha-2b every other day intramuscularly, and 300 mg RBV orally three times a day. Dynamic observations were done for HCV RNA and HIV RNA loads, CD4+ and CD8+ T lymphocyte counts, liver function and blood cell measures, and the side-effects of the medicines., Results: After 12 weeks and 24 weeks of IFNalpha and RBV combination therapy, mean HCV RNA levels reduced 1.14 log (t = 3.843, P < 0.01) and 2.08 log (t =6.564, P < 0.01) from the baseline at week 0 in the HCV-HIV co-infection group, and reduced 1.48 log (t = 6.438, P less than 0.01) and 2.33 log (t = 7.343, P < 0.01) in the HCV infection group. Meanwhile, the HIV RNA levels decreased 1.22 log (t = 3.662, P < 0.01) and 1.73 log (t = 6.119, P < 0.01) from the base line. However, there were no obvious different changes among T lymphocyte counts of HCV-HIV and HCV patients at week 0, week 12 and week 24. All 27 patients showed satisfactory biochemical response to therapy. There were some mild or moderate influenza-like symptoms, intestinal discomfort and decreased blood cell counts in the early stages of the treatments. No neuropsychic and auto-immune disorders were found., Conclusions: IFNalpha-2b and RBV combination therapy showed similar anti-HCV effects during the 24 week treatment for HCV-HIV and HCV infected patients, and some anti-HIV effect was also observed. No obvious different biochemical responses and side-effects were found between the above two groups.

Objective: To explore hepatitis C virus (HCV) non-structural protein 5A (NS5A)'s influence on inhibition of AFP expression executed by p53 protein and its possible molecular mechanism., Methods: Plasmid transfection and MEIA were employed to observe p53's inhibitive effect on AFP expression of Huh7 cells and the HCV NS5A's influence on p53 function. Western blot was employed to find out if HCV NS5A affects p53 protein expression and GST pull down assay was applied to examine the interaction between HCV NS5A and p53., Results: The AFP concentration in the supernatant of the culture of the Huh7 cells transfected with pRc/CMV was (14322+/-2412) ng/ml, and that of the Huh7 cells transfected with pCNS5A was (13843+/-3218) ng/ml; no significant difference existed between these two groups (t = 1.42, P > 0.05). After transfection with pC53-NS3, the AFP level was decreased to (10 241+/-1326) ng/ml, and in comparison to the above two groups it had a statistically significant difference (t values were 2.41 and 2.38, P < 0.05). When co-transfected with pCNS5A and pC53-NS3, the AFP expression (14582+/-1238) ng/ml returned to the level of pRc/CMV transfected, and there was a remarkably significant difference between this and that of the pC53-NS3 transfected cells (t = 3.12, P < 0.01). HCV NS5A had no function on the p53 protein expression with Western blot experiment. In the GST pull down assay, an HCV NS5A protein band was found after GST-p53 was added, but not detected with GST only., Conclusion: We found that p53 has an inhibitive function on the AFP expression in Huh7 cells and HCV NS5A minimized this p53 function. HCV NS5A did not affect p53 protein expression, but was able to form a complex with p53, by which HCV NS5A inactivated this p53 function.

Aim: To evaluate the expression of apoptosis related gene Fas ligand (FasL) in human hepatocellular carcinoma (HCC) cells HepG2 and its significance in apoptosis., Methods: Levels of soluble Fas ligand (sFasL) in a group of patients with hepatitis B virus (HBV)-induced chronic hepatitis, HBV-positive liver cirrhosis and HCC were evaluated. In a further study, the recombinant eukaryotic expression plasmid pcDNA3.1hisB-FasL was transfected into HCC cells HepG2 by lipofection, and then soluble FasL was examined in the supernatant of culture cells by EIA, FasL expression in HepG2 cells was detected by immunohistochemistry. After being stained by annexin V and propidium iodine, cells were passed through a flow cytometer and examined by a fluorescence microscope and a laser scanning microscope., Results: The sFasL levels were significantly lower in patients with HCC when compared to the patients with hepatitis or liver cirrhosis. In comparison with untransfected cells, the soluble FasL could be detected in the supernatant of transfected cells. FasL was expressed on the membranes and cytoplasm of transfected cells. The apoptotic cell rate was 36.30% in transfected cells, and was 11.53% in untransfected cells. Moreover, the different stage of apoptotic cells could be distinguished by annexin V and propidium iodine staining., Conclusion: Fas ligand is an apoptotic pathway of HCC cells.

Aim: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC)., Methods: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing, and the product expressed was detected by RT-PCR and immunofluorescence methods., Results: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis, and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immunofluorescence staining., Conclusion: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma.

Objective: To explore the activity of nuclear factor kappa B (NF-kappaB) in peripheral blood lymphocytes (PBL) of patients with hepatitis B., Methods: The PBL of patients with different types of hepatitis B and healthy individuals were isolated and then the nuclear extract was prepared. Assessment of NF-kappaB DNA binding activity was performed by electrophoretic mobility shift assay (EMSA) using digoxin labeled double-stranded oligonucleotide containing kappa B consensus sequence., Results: Densitometric scanning of the EMSA bands showed that the mean optical densities (A) from the groups of normal control, acute hepatitis B, chronic hepatitis B and chronic severe hepatitis B were 20.18+/-2.16, 27.75+/-4.11, 13.90+/-3.20 and 8.02+/-2.65 respectively. Analysis of variance showed the F value was 26.112 and the difference among the groups was significant. The difference between two groups with Dunnett T3 analysis showed there are statistically difference between the groups of the normal group and the chronic severe hepatitis B group, the acute hepatitis B group and the chronic hepatitis B group, and the acute hepatitis B group and the chronic severe hepatitis B., Conclusion: The activity of NF-kappaB in PBL of patients with hepatitis B is related with the different outcomes of hepatitis B virus (HBV) infection. Decreased activity of NF-kappaB may be an important cause for the dysfunction of PBL in chronic HBV-infected patients.

Objective: To study the inhibition effect of HCV NS5A on p53 protein transactivity and its possible mechanism., Methods: Luciferase reporter gene system was used for the study of p53 transactivity on p21 promoter and electrophorectic mobility-shift assay (EMSA) was applied to observe whether HCV NS5A could suppress the binding ability of p53 protein to its specific DNA sequence., Results: Endogenous p53 protein could stimulate p21 promoter activity, and the relative luciferase activity increased significantly (3.49 x 10(5) vs 0.60 x 10(5), t = 5.92, P<0.01). Exogenous p53 protein also up-regulated p21 promoter driving luciferase expression, comparing to the control group (0.47 x 10(5)), the relative luciferase activity increased (5.63 x 10(5)) obviously (t = 10.12, P<0.01). HCV NS5A protein inhibited both endogenous and exogenous p53 transactivity on p21 promoter in a dose-dependent manner (F > or = 20.71, P<0.01). In the experiment of EMSA, p53 could bind to its specific DNA sequence, but when co-transfected with HCV NS5A expressing vector, the p53 binding affinity to its DNA decreased., Conclusion: HCV NS5A can inhibit p53 protein transactivity on p21 promoter through its inhibiting of p53 binding ability to the specific DNA sequence.