Objective To evaluate the effect of silencing leptin by small interfering RNA(siRNA)on the expression of lep‐tin ,and apoptosis ,proliferation and intracellular Ca2+ concentration([Ca2+ ]i )of hepatic stellate cells(HSCs)and to provide evi‐dence for liver fibrosis gene therapy.Methods HSCs were divided into normal control group ,blank vector group ,siRNA nega‐tive control group and leptin‐siRNA group.After transfection of the leptin‐siRNAs into HSCs ,cell proliferation was measured by MTT assay.Cell cycle and apoptosis were measured by flow cytometry.Expression of leptin was detected by immunocyto‐chemistry and Western blot. [Ca2+ ]i was measured by Fura‐2/AM loading.Results Compared with the normal control group , the blank vector group and the siRNA control group ,the protein expression of leptin and the cell growth were significantly in‐hibited in the leptin‐siRNA group(P<0.05). The proliferation rate of HSCs was significantly different at different time points (24 ,48 and 72 h)(P<0.05).The cell apoptosis rate was increased significantly in the leptin‐siRNA group(P<0.01).At the same time ,Leptin‐siRNA‐induced [Ca2+ ]i was also significantly reduced(P<0.05).Conclusion The leptin gene may play an important role in liver fibrosis progression and is potentially a novel predictive and prognostic marker for liver fibrosis.

Since HBV DNA integration was discovered for the first time in 1980, various methods have been used to detect and study it, such as Southern Blot, in situ hybridization, polymerase chain reaction and so on. HBV DNA integration is thought to be random on the whole although some hot spots of integration were described by some researchers, one of which might be the repetitive sequences of the genomic DNA. Besides, DNA damage, especially double-strand breaks could promote HBV DNA integration into host genome. HBV DNA integration into cells may damage the stability of the genome, cause DNA rearrangement, promote DNA deletion and induce the formation of HCC.

Objective The newly developing gene therapy method and dominant negative mutants were bein g used as new promising HBV therapy method, and a dominant negative mutant of HB V X g ene we have reported in our previous report has some effects both on HBV replica tion and expression in transient expression, but the effects were interfered by persistent secretion of HBV in HepG 2 2.2.15 cell line in the experiment. To mak e sure the effects of dominant negative mutant of HBx gene, we established a HBx DN stably expressing cell clone, and evaluated the effects of HBx dominant negat ive mutant on HBV gene expression. Methods The prev HBx-GFP dominant mutant and the plasmids pRev Xwt, pRev GFP which contain the wild type X gene or GFP gene then transfected into HepG 2 2.2.15 cells by liposome. The HBsAg, HBeAg by in media were as sayed by RIA and HBV-related RNA were assayed by Northern blot. Results The pRev HBx-GFP, GFP and wild type X constructs can be effectively expressed in HepG 2 2.2.15 cells. The stable expressed HBx -GFP can significantly reduce HBeAg, HBeAg in media and the HBV-related RNA in HepG 2 2.2.15 cells, but not for pRev Xwt and pRev GFP. Conclusions The dominant negative mutant pRev HBx-GFP can significantly inhibit the HBV gen e expression. It also suggested that X gene may be one promising target for HBV gene therapy.

Objective To construct an expression vector harboring CYP2B1 suicide gene, and detect its expressions in tumor cell lines. Methods PCR amplification was performed using primers based on murine CYP2B1 gene sequence from gene bank and pc3/2B1 as template. PCR product was directly inserted an eukaryotic expression plasmid pcDNA3.0. The recombinants were analyzed and identified by restriction enzyme analysis, PCR and sequencing. Then the recombinant vector pcDNA3.0/CYP2B1 was transfected into three tumor cell lines by liposome-mediated method. The expressions of CYP2B1 gene in all the cell lines were detected by RT-PCR method. Results pCDNA3.0/CYP2B1 vector was successfully constructed, and could express CYP2B1 mRNA in the three tumor cell lines. Conclusion Eukaryotic expression vector pcDNA3.0/CYP2B1 containing CYP2B1 gene under the control of a CMV promoter is an novel effective expression vector for tumor gene therapy.

The effects of all-trans-retinoic acid (ATRA) in low doses supplementation on concentrations of polar retinoid metabolites (PRM) and retinoids in the ethanol-fed rat liver, and on hepatocyte injury were investigated. The rat model of alcoholic liver disease (ALD) was induced by intragastric infusion of ethanol, and then the rats were administrated with ATRA in two different doses (150 microg/kg body weight and 1.5 mg/kg body weight) for 4 weeks. Concentrations of retinoids in rat liver and plasma were determined by using HPLC. Liver tissues pathologic changes were observed under the light microscopy and electron microscopy. The serum transaminases concentrations were measured. The results showed that the HPLC analysis of retinoids revealed that retinoids (vitamin A, RA, retinyl palmitate) concentrations in ethanol-fed rat liver and RA concentration in ethanol-fed rat plasma were markedly diminished (P<0.01) after ethanol feeding for 12 weeks. Furthermore, obvious peaks of PRM were formed in livers of ethanol-fed rats. ATRA 150 microg/kg supplementation in ethanol-fed rats for 4 weeks raised RA concentration in both liver and plasma, and also raised vitamin A concentration in liver to control levels, partially restored retinyl palmitate concentration (P<0.05) in liver. ATRA 1.5 mg/kg supplementation raised not only RA concentrations in liver and plasma but also retinyl palmitate concentrations in liver. However, the vitamin A concentration in liver of ATRA-supplemented rats (1.5 mg/kg) was higher than that of controls (P<0.05). The histologic observation of liver tissues indicated that ATRA treatment notably alleviated hepatocellular swelling, steatosis, the swelling of mitochondria and proliferation of smooth endoplasmic reticulum (SER). ATRA treatment greatly decreased levels of serum transaminases as compared with the only ethanol-fed group (P<0.05). It was concluded that low-dose ATRA treatment could restore retinoids concentrations and abolish the PRM formation in liver of ALD rats, and then ameliorate the injury of liver cells.

A novel HBV integration site involved in hepatocarcinogenesis was investigated. The HBV DNA integration sites were detected by Alu-PCR in hepatocellular carcinoma tissues, matched surrounding liver tissues in 30 patients with hepatitis B-related hepatocellular carcinoma (HCC) and 3 cases of normal liver tissues. The integration sites and flanking sequences in human genome were sequenced and blasted, and the expression of integrated HBV genes was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The influence of the up-regulated expression of integrated genes on hepatocarcinogenesis was analyzed. Nineteen integration sites of HBV DNA into HCC tissues were obtained by RT-PCR and sequencing. These genes encoding proteins were: LOC51030, LOC157777, minichromosome maintenance complex component 3 associated protein (MCM3AP), MCTP1, SH3 and multiple ankyrin repeat domains 2 isoform 2, CCDC40, similar to HCG2033532, mitochondrial ribosomal S5 pseudogene 4. One of them was integrated into the intron of MCM3AP. RT-PCR demonstrated that the expression levels of MCM3AP mRNA in HCC tissues, matched surrounding liver tissues and normal liver tissues were in a descendent order. The ratio of MCM3AP mRNA to the GAPDH mRNA in these three tissues was 1.07375, 0.21573, 0.06747 respectively, with the difference being statistically significant among them (P<0.05). Meanwhile, the expression levels of MCM3AP mRNA from HCC tissues in which HBV DNA integrated into MCM3AP were still significantly higher than those without HBV DNA integrated into MCM3AP. It was concluded that the HBV DNA integration sites into human genome were random, and MCM3AP was a new site. The up-regulated MCM3AP mRNA may affect flanking sequences which promote the hepatocarcinogenesis.

Objective To study the development a clinical useful microarray which can detect 4 most commonly and well documented sites for detection of lamivudine-resistance mutants in HBV lamivudine therapy. Methods (1) The selected 16 oligonucleotide probes to screen 4 mutated sites in YMDD region are immoblized in a specific treatment slice by GMS 417 Arrayer. The target 308 bp nucleic acids segment of HBV were amplificated and labeled with Cy5-dCTP fluorescent dye by PCR. After hybridization of target DNA with microarray, the microchips were scanned with Gene TAC LS IV scanner and the data were obtained after processed in computer. (2) To evaluated the specialty and reproducibility of the microarray, the plasmid with Leu515Met, Met539Ile, Met539Val,V542I mutations were condstructed and serve as positive sample. Another 50 sera of lamivudine treated Hepatitis B patients for 6 to 12 months as well as 50 sera without lamivudine administration were assayed by this microarray to evaluated the rate and genetype in lamivudine related resistance mutation. Results The microarray can identify a single base change of selected lamivudine resistance-related mutation and multiple mutation detection by a single assay as well. The specialty are well in agreement with sequencing for 98%. The reproducibility rate of repeat examination is range from 96%-100%. Conclusion The microarray of lamivuine resistance related mutation can identify a single base change as multiple mutations well. And its hight reproducible results may be useful for clinical monitoring lamivudine resistance related HBV mutation.

Objective To prepare and identify rabbit polyclonal antibody against embryonic liver fordrin 3(ELF3),and investigate the distribution of ELF3 in mice tissue.Methods ELF3 specific N-terminal peptide was synthesized,and conjugated to Keyhole limpet hemocyanin(KLH)as immunogen.The ELF3-KLH complex was injected into rabbits subcutaneously,and then ELF3 antibody was purified using affinity chromatography.The titer of the antibody was evaluated by ELISA.The specificity of antibody against ELF3 and immunolocalization of ELF3 were evaluated by using Western blot and immunohistochemistry.Results Rabbit polyclonal antibody against ELF3 was prepared by the immunization of ELF3-KLH complex.ELISA and Western blot results showed the antibody against ELF3 had high titer and specificity.Western blot and immunohistochemical studies demonstrated ELF3 was expressed in the mouse heart,liver,brain and kidney tissue,particular on the cell membrane.Conclusion The preparation of polyclonal antibody against ELF3 was successful due to its high titer and specificity;ELF3 was expressed in the mice heart,liver,and kidney,particular on the cell membrane.It will provide an excellent tool for further study on the ELF3 function.

HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain active polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombinant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Recombinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were infected with the recombinant virus containing HBV polymerase gene to express the target protein. HBV polymerase expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.

The effects of PEG10 on hydrogen peroxide (H(2)O(2))-induced apoptosis in human normal liver cell line L0(2) were investigated. The PEG10 gene was transfected into L0(2) cells by lipofectamine, the positive clone was screened by G418 and defined as L0(2)/PEG10, while the cell transfected with empty expression vector (pEGFP-N1) was defined as L0(2)/vector. L0(2)/vector and parental L0(2) cells served as control. RT-PCR and Western blotting were employed to detect the expression of target genes. H(2)O(2) (50-400 mmol/L) was administered to induce the apoptosis of L0(2) cells. Cells viability was measured by MTT and the morphological changes of apoptotic cells were determined by fluorescence microscopy using hoechst33342 nuclei staining. DNA fragmentation was observed by agarose gel electrophoresis. PEG10 mRNA and protein levels in L0(2)/PEG10 cells were significantly increased as compared with those in the control cells. After treatment with 400 mmol/L H(2)O(2) for 24 h, the cellular growth inhibition rate of L0(2)/PEG10 cells was significantly lower (58.2%) than that of L0(2) (92.5%) and L0(2)/vector (88%). Distinct morphological changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were not observed in L0(2)/PEG10. Ladder-like DNA fragmentation in a dose-dependent manner was observed in both L0(2) and L0(2)/vector cell lines, but not in L0(2)/PEG10. PEG10 over-expression significantly inhibited cytotoxicity induced by H2O2 on human normal liver cell line L0(2) by antagonizing H(2)O(2)-induced apoptosis.