0.05). Serum glutamate pyruvate transaminase activity significantly decreased in the injured liver BMSCs group (P

OBJECTIVETo construct a eukaryotic expression vector to express the hepatitis E virus protein open reading frame 3 (ORF3) and investigate the intracellular location of the expressed protein using the baby hamster kidney (BHK-21) fibroblast cell line.

METHODSThe ORF3 gene was amplified by RT-PCR, cloned into the HindIII and EcoRI sites in the multicloning site of the pDsRed-Monomer-N1mammalian expression vector that encodes a red fluorescent protein (DsRed), and confirmed by restriction enzyme digestion and sequencing. The recombinant plasmid was then transfected into BHK-21 cells via the Lipofectamine 2000 reagent; the subsequent ORF3 gene overexpression was confirmed by RT-PCR and the protein expression and location was detected by Western blotting and immunofluorescence assay.Results TThe pDsRed-Monomer-N1-ORF3 recombinant plasmid was successfully constructed. After transfection into BHK-21 ceils, the ORF3 gene was transcribed and expressed, and the ORF3 protein was mainly located in the cytoplasm, where it could react with a specific antibody.

CONCLUSIONThe ORF3-DsRed fusion protein was mainly located in the cytoplasm of BHK-21 fibroblasts, and may represent a useful tool for research on the role of this protein in HEV infection.

Animals ; Cell Line ; Cricetinae ; Cytoplasm ; Fibroblasts ; metabolism ; Genetic Vectors ; genetics ; Hepatitis E virus ; metabolism ; Luminescent Proteins ; Open Reading Frames ; Plasmids ; Recombinant Proteins ; Transfection ; Viral Proteins ; metabolism

[Objective]People infected with Hepatitis B are often divided into hepatitis B carriers and hepatitis B patients based on whether ALT is normal or not,and ALT ≥ 2UNL is one of the indications for clinical antiviral treatment,but no sufficient evidence to justify this. In order to explore the theoretical basis,the paper investigated the effects of hepatitis B virus(HBV) on hepatic stellate cells(HSCs).[Methods]A total of 132 chronic hepatitis B patients with different viral loads and ALT levels were randomly selected as the study subjects. Of these patients,those with ALT≥2UNL were treated with antiviral therapy and followed up for 24 weeks. The effects of HBV on HSCs before and after the treatment were compared and analyzed. HSCs proliferation was detected by MTT method,HSCs cell cycle by flow cytometry,and expression of TGF-β1,Smad3,Smad7,α-SMA,collagen Ⅰ,collgen Ⅲ mRNAs and corresponding proteins by RT-PCR and Western blotting,respectively.[Results]At the normal ALT level,HBV with different viral loads had no significant effect on the proliferation,cell cycle and cell secretion of the HSCs. At the abnormal ALT level,especially when ALT ≥ 2UNL,with the increase of virus loads,HSCs proliferation accelerated;cells in the G0/G1 phase decreased significantly and cells in the S and G2/M phases increased significantly;the expression of TGF-β1,Smad3,α-SMA,collagen Ⅰ,collgen Ⅲ mRNAs and corresponding proteins increased significantly,but Smad7 mRNA and protein expression decreased significantly,the differences were statistically significant. HBV showed a significantly lower effect on HSCs after the antiviral therapy than before.[Conclusions]This paper reveals the differential effects of HBV on HSCs at different ALT levels and presents a comparative analysis of the effects before and after the antiviral therapy,which provides a theroretical basis for identifying the ALT level as an indication for HBV antiviral therapy.