To construct a subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus with suppression subtractive hybridization technique, the mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-) NS5A and pcDNA3 1(-) empty vector, respectively, then the cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNAs were obtained. Then the tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After the tester cDNA was hybridized with the driver cDNA twice and underwent two times of nested PCR, it was then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. The amplified library contained 121 positive clones. Colony PCR showed that 115 clones contained 200- 1 000 bp inserts. Sequence analysis was performed in 90 clones, and the full length sequences were obtained with bioinformatics method. Altogether 46 kinds of coding sequences were acquired, which consisted of 31 kinds of known and 15 kinds of unknown ones. The obtained sequences might be target genes transactivated by NS5A protein of HCV, among which some genes coding proteins involved in cell cycle regulation, cell apoptosis, signal transduction pathway and tumour development. The results indicated that the subtractive library of genes transactivated by NS5A protein of HCV was constructed successfully, which brought some new clues for studying the biological functions and pathogenesis of the viral proteins

To investigate the potential role of hepatitis B virus core protein (HBcAg) in the pathogenesis of hepatitis B and seek for an effective therapeatic approach to the treatment of hepatitis B, the improved yeast two hybrid technique was employed to construct HBcAg bait plasmid, and the plasmids were transformed into yeast AH109. Then the yeast AH109 was mated with yeast Y187 containing liver cDNA library plasmid in 2?YPDA medium, diploid yeast was plated on both synthetic dropout nutrient medium (SD/ Trp Leu His Ade) and synthetic dropout nutrient medium (SD/ Trp Leu His Ade) containing X ? gal to be selected and screened two times. Plasmids were extracted from sixteen positive colonies, and they were sequenced. Among them, two colonies contained metallothionein. The interaction between HBcAg and metallothionein was confirmed by in vitro reticulocyte lysate translocation and immunoprecipitation. The results suggested that the pathogenesis of hepatitis B metallothionein might play an important role in the episode process.

Objective To study the changes in PAF and its receptor in Kupffer cells in experimental cirrhosis and to evaluate the role of activated Kupffer cells in portal hypertension. Methods Kupffer cells, isolated from the livers of control and CCl_4-induced cirrhotic rats, were cultured in serum-free medium overnight. PAF synthesis and release by Kupffer cells were determined 24 h later by rapid ~3H-PAF scintillation proximity assay, and the expression of PAF receptor in Kupffer cells by saturation binding technique and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). By immunohistochemisty, the distribution of PAF in Kupffer cells was surveyed. Results Cell-associated PAF synthesis and release was increased about 1.48 fold and 2 fold, respectively, by Kupffer cells in cirrhotic liver as compared with the control (P0.05). Consistent with the receptor binding capacity, the mRNA expression of PAF receptor increased significantly in the Kupffer cells of cirrhotic liver (P

Objective To investigate the correlation between Kupffer cells and the synthesis of platelet activating factor(PAF)in experimental hepatic cirrhosis,and to elucidate the effects of endothelin(ET)on portal hypertension.Methods Thirty SD rats were randomly assigned to two groups:control group and CCl4-induced hepatic cirrhotic group.Kupffer cells,isolated from the livers of animals in both groups,were cultured for 24h.ET-1-induced PAF synthesis,and mRNA expression of PAF,ET-1 receptor and preproendothelin-1 in Kupffer cells were determined by rapid 3H-PAF scintillation proximity assay,saturation binding technique and semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR),respectively.Results Cell-associated PAF synthesis and release increased about 1.48 folds and two-folds,respectively,by cirrhotic Kupffer cells as compared to the control(1.02 ? 0.06 vs 0.69 ? 0.07 pg /mg DNA in Kupffer cells and 1.42? 0.14 vs 0.66 ? 0.04 pg/mg DNA in medium).Endothelin-1 enhanced Kupffer cells to stimulate PAF synthesis in a concentration-dependent manner,and for cirrhotic Kupffer cells,the effect was more significant than control.Cirrhotic Kupffer cells also had increased densities of functional receptors for both PAF and ET-1(exclusively ETB),but did not change the affinity of these receptors.No mRNA transcripts for the ETA receptor or preproET-1 were detected.Conclusion Kupffer cell is the main source of PAF in the cirrhotic rats.ET-1 stimulates PAF synthesis in activated Kupffer cells via ETB receptor.Since both ET-1 and PAF individually cause portal hypertension,Kupffer cells may play a role in portal hypertension associated with liver cirrhosis.

To investigate the potential role of hepatitis B virus(HBV) preS1 protein in mediating HBV adhesion to liver cell, we prepared recombinant proteins of HBV preS1 in yeast. PCR was performed to amplify the gene of HBV preS1 from the plasmid pCP10/HBV ayw subtype containing the whole fragment of HBV and the PCR product was cloned into pGEM T vector. The gene of HBV preS1 was cut from pGEM T vector and cloned into yeast expression plasmid pGBKT7, the pGBKT7 plamids containing preSl were transformed into yeast cell AH109. The yeast protein was isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting. The results showed that the presence of HBV presl proteins in yeast cells was confirmed by Western slot analysis. the molecular weight of the expressed product was about 30000 Da. The findings indicated that HBV preS1 was successfully expressed in yeast system.

Interleukin 18 (IL 18) is emerging as a powerful, pleiotropic cytokine involved in determining the polarization of T cell responses. To identify the effect of IL 18 on hepatitis B virus, mouse IL 18 gene was transferred and expressed in 2.2.15 cells. Meanwhile, the inhibition of HBsAg, HBeAg and HBV DNA was investigated. Reverse transcription polymerase chain reaction (RT PCR) was used to amplify the mouse IL 18 from spleen cell of Balb/c mouse challenged with lipopolysaccharide (LPS) and phytahematoagglutinin (PHA), and reconstruct plasmid pLXSN IL18 with the retroviral vector pLXSN. Both pLXSN IL18 and pLXSN were transfected into PA317 cells and then 2.2.15 cells were infected by using the supernatant containing pseudovirus released by PA317. HBsAg and HBeAg were detected by ELISA and the HBV DNA was determined by quantitative PCR. The 580bp of mIL 18 was cloned into retroviral vector pLXSN. The pseudovirus contained in the supernatant of transfected PA317 cells was identified by RT PCR. When the pseudovirus was infected into 2.2.15 cells and incubated for 3, 5, 7, 14 days, the P/N value of HBsAg and HBeAg decreased gradually and became negative on day 14. The copies of HBV DNA reduced markedly. The results indicate that mIL 18 gene expressed in 2.2.15 cells can significantly inhibit the replication expression of HBV and may be used as a potential agent for gene therapy against HBV infection.

Objective To evaluate the therapeutic effect,safety and complication of percutaneous argon-helium cryoablation in the treatment of primary hepatocellular cancer(HCC).Methods Three hundred HCC patients were treated with percutaneous argon-helium cryoablation under ultrasound guidance with argon-helium cryosurgical system.Results Two hundred and thirty-three tumors(diameter ranged from 5.0cm to 15cm,with a mean of 7.2cm?2.8cm),in 165 patients were considered to be incompletely ablated,while 185 tumors(diameter ranged from 1.9cm to 7.0cm with mean value of 5.6cm?0.8cm) in 135 patients were completely ablated.There was a significant difference in tumor diameter between these two groups(P

To investigate the biological functions of hepatitis B virus e antigen (HBeAg) on pathogenesis of HBV, and to seek for effective methods to prevent and cure hepatitis B, we performed the yeast two hybrid system 3 technique to construct HBeAg bait plasmid. The bait plasmid was transformed into yeast AH109 and expressed in it. After the expression of HBeAg was identified by SDS page and Western blot, the AH109 yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2 YPDA medium to form diploid yeast and plated on synthetic dropout nutrient medium (SD/ Trp Leu His Ade) and synthetic dropout nutrient medium (SD/ Trp Leu His Ade) containing x ? gal for screening. Plasmids of blue colonies were extracted and transformed into Escherichia coli, then analyzed by DNA sequencing and bioinformatics. Thirty nine positive colonies were sequenced, among them three colonies were metallothionein. To further prove the interaction between HBeAg and metallothionein, translation was performed by using reticulocyte lysate, and immunoprecipitation was showed in vitro . The results indicated that hepatitis B virus e antigen could interact with metallothionein in vivo and in vitro.

Objective To investigate the biological functions of hepatitis B virus e antigen (HBeAg) protein. Methods Yeast-two hybrid technique was performed to look for proteins interacting with HBeAg from hepatocytes. HBeAg bait plasmid was constructed by cloning the HBeAg gene into carrier plasmid pGBKT7, then the yeast AH109 (a type) was transformed. The transformed yeast cells was amplified and mated with yeast cells Y187 (?type) containing liver cDNA library plasmid pCAT2 in 2?YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium and were selected for two times. Results Plasmids of positive colonies were extracted and analyzed by DNA sequencing and BLAST in GenBank. Conclusions After the full sequence of new gene E-36 was amplified from the mRNA of HepG2 cells by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pGADT7 vector, the recombinant plasmid was translated by reticulocyte lysate and analyzed by immuno-coprecipitation technique in vitro together with HBeAg. The result provided some new clues for the study of the biological functions of HBeAg.

Bile acid metabolism, gut microbiota, and bile acid receptors are involved in the development and progression of hepatocellular carcinoma (HCC). There are substantial increases in the levels of some bile acids, such as glycocholic acid, taurocholic acid, and taurochenodeoxycholic acid, in the liver tissue of HCC mice and the serum and feces of HCC patients. Bile acid metabolism due to the imbalance of the abundance of bacteria producing bile salt hydrolases and Clostridium in the intestine and the change in immune microenvironment may also promote the development of HCC. Moreover, some bile acid receptors, such as farnesoid X receptor, G protein-coupled bile acid receptor 1, pregnane X receptor, constitutive androstane receptor, and sphingosine-1-phosphate receptor 2, have been shown to participate in the development and progression of HCC through various pathways. Each link of bile acid metabolism plays a different role in the progression of HCC, and a systematic elaboration of the interaction between these links may help to deepen the understanding of the pathogenesis of HCC and develop the biological targets for early diagnosis, prognosis prediction, and precise treatment.