The Effect Of L02Transfected With Hepatitis C Virus NS2Gene On Macrophages In Liver Environment

Objective: Hepatitis C virus (HCV) was discovered in1989by Choo etc.About170million people were infected worldwide until now, accounting for2-3%of the world ’s population. Anti-HCV positive incidence is3.2%inhealthy populations of china. HCV not only lead to chronic infection but alsocause fibrosis, cirrhosis, and hepatocellular carcinoma.HCV is a single positive strand RNA virus andencode a polyproteinwhich is cleaved by proteases of host and virus into ten distinct proteins:structural proteins (Core, E1, E2, p7) and non-structural proteins (NS2, NS3,NS4A, NS4B, NS5A, and NS5B). NS2protein, as a key part of the connectionof HCV structural proteins and non-structural proteins, has been a researchfocus. NS2not only play an important role in the assembly and release ofHCV virus particles, is also involved in regulation of gene expression andapoptosis of the host cell. Erdtmann found that NS2interacted with theC-terminal region of CIDE-B to inhibit CIDE-B-induced apoptosis. NS2alsocan inhibit promoter activity of IFN-βgene in host cells. In the study, fullNS2gene was acquired from JFH-1and constructed eukaryotic expressionplasmid pEGFP-C1-NS2. Subsequently, the proliferation and secretedcytokines of L02cell line of stable expression pEGFP-C1-NS2were observed.In the other word, the effect of NS2on host cells will be elucidated.There aremany innate immune cells in the liver, including kupffer cells and NK cellsetc,which play an important role in anti-viral infection. But in the livermicroenvironment, HCV proteins may interfere the signal transduction ofimmune cells and inhibite the immune response,which account for thepersistence of HCV. In this study, using transwell co-culture system, thechangese of macrophages were detected which were co-cultured with L02stably transfected with pEGFP-C1-NS2or pEGFP-C1-vector. It described a novel way about the pathogenesis of hepatocellular carcinoma and themechanisms of immune escape of HCV.Methods：1HCV NS2gene fragment was obtained from full gene plasmid of JFH-1strain (HCV2a) by PCR with designed primers.The PCR product was insertedinto pMD18-T vector and sequenced. The positive recombinant plasmid andthe eukaryotic expression plasmid pEGFP-C1were cut with the sameenzymes, BamHI and HindⅢ. Thus a recombinant eukaryotic expressionplasmid pEGFP-C1-NS2was constructed.2The plasmid pEGFP-C1-vector and pEGFP-C1-NS2was respectivelytransfected into L02cells with Lipofectamine2000transfection reagent. Theexpression of NS2protein was detected by Western Blot.3The proliferation of L02, L02(pEGFP-C1-vector) andL02(pEGFP-C1-NS2) cells were respectively analyzed by MTS method atdifferent time points (24h,48h,72h).4Culture L02, L02(pEGFP-C1-vector) and L02(pEGFP-C1-NS2) cells,and detecte cytokines IL-8,IL-10,TGF-β for the mRNA level changes.5The activity of THP1cells were detected, which respectivelyco-cultured with human liver cell line L02, L02(pEGFP-C1-vector) andL02(pEGFP-C1-NS2) using tanswell cell culture system. The mRNA levelchanges of proinflammatory and anti-inflammatory cytokines (IL-1β, IL-8,IL-10, TGF-β) were detected by Real-time PCR at6h and10h; inflammatorycytokines were detected by CBA at12h and24h; cellular transcription factorsSTAT3and phosphorylated STAT3were detected at12h and24h by Westernblot.Results：1A eukaryotic expression plasmid pEGFP-C1-NS2was successfullyconstructed. Agarose gel electrophoresis analysis of pEGFP-C1-NS2diegested with BamHI and HindⅢ show that there is a650bp fragment.2L02cells transfected with pEGFP-C1-vector and pEGFP-C1-NS2weredetected by Western blot, show that a single band protein of GFP-NS2fusion protein are observed by anti-GFP monoclonal antibody.3L02cell proliferation curve shows that L02(pEGFP-C1-NS2)cellscompared to L02and L02(pEGFP-C1-vector)cells on the proliferation was notobvious.（P>0.05）4IL-10,TGF-β mRNA levels of L02(pEGFP-C1-NS2) cells weresignificantly higher than other groups (P<0.05). IL-8mRNA levels ofL02(pEGFP-C1-NS2) cells was not obvious than L02(pEGFP-C1-vector)cells (P>0.05).5Real-time PCR results shown that IL-1β, IL-8, TGF-β mRNA levels ofTHP1co-cultured with L02(pEGFP-C1-NS2) for6h were significantly higherthan other groups (P<0.05), IL-10was not obvious than other groups(P>0.05);but IL-10mRNA levels of THP1co-cultured withL02(pEGFP-C1-NS2) for10h was significantly higher than other groups(P<0.05).6The results of CBA shown that IL-6,IL-1β,TNF,IL-10which secretedby THP1co-cultured with L02(pEGFP-C1-NS2) for12h and24h insupernatant were significantly higher than other groups (P<0.05).7THP1cells respectively co-cultured with human liver cell line L02,L02(pEGFP-C1-vector) and L02(pEGFP-C1-NS2) using tanswell cell culturesystem. The expression of phosphorylated STAT3were no obvious change inthese groups.Conclusions:1A eukaryotic expression plasmid pEGFP-C1-NS2is successfullyconstructed. The L02cells strain stably transfected pEGFP-C1-vector orpEGFP-C1-NS2were acquired.2The expression of IL-10,TGF-β in the mRNA level of L02cellstransfected with pEGFP-C1-NS2increased.3The expression of proinflammatory cytokines(IL-1β,IL-6,IL-8) andanti-inflammatory cytokines (IL-10,TGF-β) in the mRNA and protein level ofTHP1co-cultured with L02(pEGFP-C1-NS2) cells increased.