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Objective To investigate the effects of tumor-necrosis factor α (TNF-α) on the expressions of proprotein convertases (PC) and its relationship with the inhibition of hepatitis B virus (HBV) replication.Methods HepG2.2.15 cells cultured routinely were exposed to 20 μg/L recombinant TNF-α and/or 20 μmol/L PC inhibitor (DEC) for 18 h.Then Followed cells werecollected and cell total RNA and HBV DNA were extracted.PC mRNA and core-associated HBV DNA were measured using real-time polymerase chain reaction (PCR) techniques.Measurement data was compared using t-test.Results When PC mRNA expressions in the blank group was as to 1,the expressions of PC1/3、PC2、furin、PC4 、PC5/6 、PACE4 and PC7/8 mRNA in HepG2.2.15 cells treated with 20μg/L TNF-α treatment for 18 h were all up-regulated,which were 3.3±0.7、79.3±3.3、77.5±1.3、19.2±3.1、1.3±0.1、1.4± 0.2、274.8± 7.1,respectively (all P＜0.05).Treatment of 20 μg/L recombinant TNF-α for 18 h significantly reduced core-associated HBV DNA compared with blank gourp (0.21∶1,t =8.79,P =0.002),while 20 μmol/L DEC significantly up-regulated core-associated HBV DNA (3.84∶ 1,t=7.67,P=0.004).Moreover,core-associated HBV DNA in group of DEC and TNF-α treatment was significantly higher than group of TNF-α treatment (0.31∶0.21,t=10.49,P=0.007).Conclusion Up-regulated PC mRNA expression induced by TNF-α is significantly associated with the inhibition of HBV replication.
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Tandem-repeated sequence of nucleic acid was constructed by splicing 4 fragments which contain the same sequence in the central part, using overlap extension polymerase chain reaction and then repeatedly cloning it in the same vector at different site of restriction endonuclease. Its signal amplification action was evaluated using electrophoresis of hybridized product and dot hybridization assay. 24-repeat sequence was successfully constructed and confirmed by restriction endonuclease digestion analysis. The construct was 25-repeat actually since the vector itself had the same basic sequence. Hybridized product electrophoresis revealed that the 25-repeat sequence could combine with several secondary probes. Dot hybridization assay demonstrated that tandem-repeated sequence was 16-fold more sensitive than that of non-repeated sequence. Tandem-repeated sequence had good effect on signal amplification. It could be easily cheaply prepared in large amount after cloning. Thus, it might be useful in clinical examinations and biological researches.
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Genetic Vectors , Nucleic Acid Hybridization , Methods , Polymerase Chain Reaction , Methods , Tandem Repeat Sequences , GeneticsABSTRACT
AIM:To study the effect of proprotein convertases (PCs) on the transforming growth factor (TGF) ?1-induced inhibition of HBV replication.METHODS:HepG2.2.15 cells cultured regularly were exposed to recombinant TGF?1 at concentration of 2 ?g/L or 5 ?g/L and/or PC inhibitor at concentration of 20 ?mol/L for 18 h.The total RNA and HBV core particle DNA were extracted from these cells,and PC mRNA and core-associated HBV DNA were detected by real-time PCR technique.RESULTS:The mRNA expression levels of 7 PCs in HepG2.2.15 cells were observed with various degrees.Recombinant TGF?1 significantly up-regulated the mRNA expression of all PCs except for the down-regulation of PC5 /6,though PC1 /3 and PC2 were up-regulated most obviously.Furin and PACE4 were the predominant PCs before and after TGF?1 exposure when the basic mRNA expression was taken into account.Further study showed that TGF?1-induced the inhibition of HBV replication was abrogated by PC inhibitor in HepG2.2.15 cells.CONCLUSION:TGF?1-induced the inhibition of HBV replication is mediated by the up-regulation of PCs,which might be of many implications in efficient interferences of TGF?1 on HBV replication.
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Objective To study the SEN-V DNA in liver tissue from patients with non A to E hepatitis in Guangzhou area. Methods A set of primers according to the original sequence from GenBank were designed , and SEN-V DNA sequence was amplified by in situ-PCR in liver tissue. Results Two of 6 patients with non A to E hepatitis were found to be SEN-V-DNA positive in liver tissue. While 8 patients with HBV infection and 6 of other liver diseases including 2 of autoimmune liver disease, 1 of Dubin Johnson syndrome, 1 of fatty liver, 1 of the primary biliary cirrhosis and 1 of drug induced hepatitis were all SEN-V DNA negative. SEN-V DNA were mainly distributed in liver cell cytoplasm, partially in nucleus. There presented the phenomenon of cell fuse. SEN-V positive cells accounted for about 3%～10% of total cells.Conclusions SEN-V can be found in liver tissue of patients with non A to E hepatitis in south China. Maybe it can cause liver damage.
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Objective To illustrate the relationship between the serum viral load, clinical types and the G1896A in precore, A1762T/G1764A in basal core promoter (BCP) or united mutation of them of HBV from patients with HBeAg-negative hepatitis. Methods Sera from 240 HBeAg-negative, 60 HBeAg-positive patients, and 40 negative controls were collected. Mutations of G1896A and A1762T/G1764A were detected using CD-PCR. Viral load in sera was demonstrated using real-time quantification PCR. Results The mutations of G1896A were detected in 57.6% and 6.7% in HBeAg-negative and HBeAg-positive patients respectively. The mutations of A1762T/G1764A were detected in 37.9% and 31.7% in the same patients respectively. The united mutations were detected in 13.5% of HBeAg-negative patients. G1896A was associated with low viral load, and A1762T/G1764A did not have any special relation viral load in sera from HBeAg-negative patients. The united mutations often occurred in patients with severe chronic hepatitis B, and been not related to the viral load. Conclusions The replication capability of G1896A variant decreased without HBeAg production as usual. For A1762T/G1764A variant, the situation of its replication activity was complicated. Its HBeAg production was less influenced than that of G1896A variant. The united variant might has higher pathogenicity and replication activity, and to which much attention should be paid in the future.
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AIM: To study the influences of P1 promoter activity of furin gene on the functions of hepatocytes in patients with liver cirrhosis.METHODS: The patients with liver cirrhosis of 180 cases were recruited.The single nucleotide polymorphism(SNP-229 C/T) in P1 promoter of furin gene was genotyped using competitively differentiated polymerase chain reaction.The relationships between the promoter activity based on genotyping and the serum levels of liver enzymes,total bilirubin,albumin and prothrombin were observed.RESULTS: The distribution frequencies of allele C and T were 75.3%(271/360) and 24.7%(89/360).Those of genotypes CC,CT and TT were 62.2%(112/180),26.1%(47/180) and 11.7%(21/180),respectively.The distribution frequencies of the genotypes were not related to the serum levels of major liver enzymes,albumin,total bilirubin and prothrombin,except for alkaline phosphatase and ?-glutamyl transferase.CONCLUSION: The activity of furin promoter exerts no effects on the main functions of hepatocytes,suggesting that furin may be a new therapeutic target for HBV infection.