| Hepatitis C virus (HCV) is a serious and growing threat to human public health.And it is estimated to infect over170million people worldwide. It has become clearthat75%to85%of persons who develop acute hepatitis C will remain HCV-infected.A high proportion of cases with chronic HCV infection often results in liver disease,including variable degrees of hepatic inflammation and fibrosis, cirrhosis, andoccasionally hepatocellular carcinoma (HCC) and, therefore, likely to be responsiblefor causing death.HCV, the principal cause of parenteral non-A, non-B hepatitis, is an positivesingle-strand RNA virus and a member of the Flaviviridae family, containing a9.6kbgenome. The genome includes a long open reading frame (ORF) that encodes apolyprotein about3,000amino acids. This polyprotein is proteolytically cleaved byviral and cellular proteases to generate at least10gene products: four structuralproteins (core, E1, E2, and p7) and at least six nonstructural proteins (NS2, NS3,NS4A, NS4B, NS5A, and NS5B). In addition to the above10gene products, a newHCV protein, termed alternate reading frame protein (ARFP/core+1protein/Fprotein), has been reported by various investigators. F protein is encoded from analternative+1reading frame overlapping the core genomic region. Studies haveshown that core protein can inhibit Fas or TNF-induced apoptosis and cooperate withRas to transform liver cells to the tumor phenotype and liver cancer cells, owing totrans-regulation of the activity of cytokines, such as c-myc, c-fos and Bcl-2by activation of NF-кB, which show that core protein may be an oncogene. And coreprotein has been implicated in variable degrees of hepatic inflammation and fibrosis,cirrhosis, and even hepatocellular carcinoma. The latest reports indicate that HCV Fprotein can over-activate NF-kB signal pathway, which makes HepG2-F cells able toresist TNF-a induced apoptosis. Discovery of F protein expressed from the HCV corecoding sequence challenges some properties assigned to core protein.On the one hand, the experiments explore the effect of the expression of F proteinon the cell malignant transformation; on the other hand, it is observed that the effectof the F protein both on the regulation of expression levels of anti-oncogenes Bax,oncongene Bcl-2and on apoptosis in HepG2cells, which contributed to clarify themechanism of F protein on the pathogenesis of chronic hepatitis C and developmentof HCV vaccine.Section Ⅰ Expression of hepatitis C virus1b F protein leadstransfected cell to transformation and Genesis of LiverCancerThe recombinant eukaryotic expression vector pcDNA3.1containing theintroduced F gene transfected Chang-liver cells with Lipofectamine2000reagent toobtain the recombinant strains Chang-liver-pcDNA3.1-F, uesd the recombinantstrains Chang-liver-pcDNA3.1-C expressing C protein as the control group. In thispart,G418was employed to screen recombinant strains. Then Anchorage-independentgrowth in soft agar was carried out and cells were inoculated subcutaneously in theback of nude mice, the tumors were harvested and the pathological identification wasdone after that. Transfected cell lines were obtained from experimental transfections,which showed anchorage-independent growth in soft agar; the tumors grew in nude mice inoculated with pcDNA3.1-F and pcDNA3.1-C cells after14and9days. After50days continuous observation, mice were killed and constitution structure of thetumors coincided with that of hepatocarcinoma by pathology. In conclusion, overexpression of F protein resulted in an increased proliferation rate in Chang-liver cells,as well as core protein. HCV F protein could lead to hepatocellular carcinoma in vivo. Section Ⅱ Regulation of anti-oncogene Bax and oncogenBcl-2by the expression of Hepatitis C virus1b F protein inhuman HepG2cellsFull length HCV1b F gene of HCV was amplified by PCR, introducing theplasmid pcDNA3.1-F containing HCV1b full length open reading frame as templateto obtain the recombinant eukaryotic expression plasmid pEGFP-C3-F, using HCVcore gene-containing pEGFP-C3-C as the control group. Respectively, HepG2cellswere transfected with plasmid pEGFP-C3-F/pEGFP-C3-C by Lipofectamine2000and were screened by the use of G418. Flow cytometry detection was used toinvestigate the effect of F protein on HepG2cells cycle. Western blotting was used todetermine the changes of the translation levels of Bcl-2and Bax genes. HCV Fprotein was found stably expressed in the recombinant plasmid. It could promoteproliferation and suppress apoptosis of HepG2cells. Expression levels of Bcl-2andBax were significantly higher in HepG2cells transfected by pEGFP-C3-F andpEGFP-C3-C than that of non-transfected cells. However, expression levels of Bcl-2and Bax in HepG2cells transfected by pEGFP-C3-F and pEGFP-C3-C, respectively, were not significantly different(P>0.05). This research revealed that F proteinpossess regulatory properties on celluar oncogen Bcl-2and anti-oncogen Bax, whichmay play an important role in the development of potentially fatal chronic hepatitis,cirrhosis, carcinogenesis. |
PDF Full Text Request