Hepatitis B Virus X Gene Induced Cell Proliferation And Upregulated Proinflamation Factors Expression Of Mesangial Cell Line Of Rat

PARTⅠConstruction of hepatitis B virus X gene eukaryoticexpression vectorObjective:To construct a highly effective eukaryotic expression vector pCI-neo withHBV X gene(pCI-neo-X).Methods:HBV DNA was extracted from the HBV DNApositive serum.HBV X gene was amplified by PCR,and identified by geloseelectrophoresis analysis.The PCR products and the eukaryotic expression vectors pCI-neowere digested by restriction endonucleases,then the digested X gene was inserted into thedigested eukaryotic expression vector.After screening,the positive recombinants werepicked out.PCR analysis and sequencing were made.Results Both vector DNA directsequence and restriction pattern confirmed that the HBV X gene was seccessfully insertedinto pCI-neo vector.Conclusion:The eukaryotic expression vextor pCI-neo has beensuccessfully constructed. PartⅡHBV X gene transfection induces glomerular mesangialcell proliferation and upregulates TNF-α,IL-1βand IL-6 expressionObjectives The aim of this study is to approach the effect of HBx on expression oftumor necrosis factor-α(TNF-α),interluekin(IL)-1β,and IL-6 in,and proliferation of ratmesangial cell of rat in vitro.Methods Liposome bearing pCI-neo-X was transfected intocultured rat mesangial cell line of the rat in vitro through liposome method.X geneexpression in the cells was assessed by Western blot.The mRNA expression of TNF-α,IL-1βand IL-6 in expression of cultured mesangial cells line was assessed bysemiquantitative RT-PCR.Mesangial cell proliferation was assessed by MTT.Results HBxobviously expressed in cultured mesangial cells line at 36^th and at 48^th hour aftertransfection.The mRNA of TNF-α,IL-1βand IL-6 expression simutanously increased.Thesupernatent TNF-αlevels in supernatents in MC group,MC plus pCI-neo and MC pluspCI-neo-X at 36^th hours after transfection were (41.7±10.3) pg/ml,(44.0±10.1) pg/ml,(185.3±70.7)pg/ml,respectively,and at 48^th hours were(43.7±10.6)pg/ml,(33.3±8.6)pg/ml,(140.3±42.1) pg/ml,respectively;The supernatent IL-1βlevels in MC group,MC plus pCI-neo and MC plus pCI-neo-X were (182±23) pg/ml,(170±18) pg/ml,(402±60) pg/ml,respectively,at 36^th,and (181±20)pg/ml,(165±19)pg/ml,(432±58)pg/ml,respectively,at 48^th;The supematent IL-6 levels in MC group,MC plus pCI-neoand MC plus pCI-neo-X were (85.7±16.9) pg/ml,(69.7±22.2) pg/ml,(177.0±18.2)pg/ml,respectively at 36^th and (70.0±18.3) pg/ml,(73.0±14.0) pg/ml,(179.7±26.3)pg/ml,respectively at 48^th,The expression of TNF-α,IL- 1βand IL-6 in pCI-neo-X groupwere all markedly higher than those in other two groups(all p＜0.05).The cell proliferationwas also obvious at the same time.Absorbance in transfcted pCI-neo-X cells were1.56±0.36 at 36^th and 1.69±0.32 at 48^th in hour after transfection,which was markedlyhigher than in untrasfected group 0.70±0.08 at 36^th,0.74±0.18 at 48^th in hour or than intransfcted pCI-neo cells 0.68±0.18 at 36^th,0.80±0.19 at 48^th in hour,respectively,all p＜0.05).Conclussion Therefore,we can conclude that HBx can induce expression ofmRNA of TNF-α,IL-1βand IL-6.HBx may play a critical role in the cell proliferationthrough TNF-α,IL-1βand IL-6 high expression.PartⅢHepatitis B virus X protein upregulates tumor necrosisfactor-αexpression of rat mesangial cell line via NF-κB and ERKspathwayObjectives HBx,a 17-kd protein encoded by X gene of HBV,has been shown tofunction as a transcriptional trans-activator of a variety of viral and cellularpromoter/enhancer elements.The aim of the study is to investigate the signal transductionpathway of HBx in glomerular mesangial cell proliferation and TNF-αexpression.Methods PCI-neo-X was transfected into cultured glomerular mesangial cells.Bothmesangial cell proliferation and supematant TNF-αexpression were compared betweentreated or untreated with ERK inhibitor U0126,NF-κB inhibitor lactacystin and p38inhibitor SB203580 respectively.HBx,ERK_1/2 and p-ERK_1/2 expression in glomerularmesangial cells was assessed by Western-blot.TNF-αmRNA expression was assessed bysemi-quantitative RT-PCR.TNF-αlevels in supernatants were measured by ELISA.Mesangial cell proliferation was assessed by MTT.Results HBx expression was found intransfected glomerular mesangial cells,and became prominent at 36 and 48 hours aftertransfection whatever with or without U0126 in culture media.TNF-αmRNA expressionwas significantly decreased in U0126 group compared with U0126-free group.TNF-αlevels in supernatants in pCI-neo-X transfection without U0126 group were (189.0±18.1)pg/ml at 36 hours and (172.3±24.3) pg/ml at 48 hours after transfection,(41.7±10.3) pg/ml.In contract,TNF-αlevels in supernatants with U0126 were (65.6±11.6) pg/ml and (84.0±24.6) pg/ml.The TNF-αlevels in the latter groups showed significantly lower thanthe formers (P＜0.05).Glomerular mesangial cell proliferation was also partially inhibitedwhen treated with ERKs inhibitor U0126 or NF-κB inhibitor lactacystin,but no effect wasobserved in p38 inhibitor SB203580 treated group at 36 and 48 hours after transfection.Conclussion HBx can induce glomerular mesangial cell proliferation and increaseTNF-αmRNA expression and its protein production.HBx upregulated TNF-αexpressionand induced cell proliferation of glomerular mesangial cell line partly through ERK_1/2signal transduction pathway.