| Objective:1.Using iTRAQ Technology,identify and quantitativelyanalysis the changes of the proteins related to HCC in patients infected withHBV. In order to obtain the HBV infection related HCC liver tissue’sdiferent proteome’s "full set of information ", and screen the molecularmarkers related to the development of HBV infection related HCC,andfurther apply the Realtime PCR and Western blot to verify from the gene andprotein expression levels. Then analysis the association of classification, andHCC clinical stage and prognosis. In order to through this study, to providenew clues and ideas for the further revealing of the pathogenesis of HCCrelated to HBV infection and looking for meaningful potential targets ofHCC marker for diagnosis and therapy.2.The selected G6PD markersspecific HCC molecules associated with HBV infection from the differencein proteome. Using HBV replication cell model HepG2.2.15by siRNA geneknockout and pcDNA3.0gene eukaryotic expression plasmid transfectiontechnology carries on the preliminary discussion on the mechanism of HBVreplication. In order to investigate the G6PD in the mechanism of HBVreplication and their use as potential value target of anti-HBV infection treatment.Methods:1The related HCC HBV infection in cancer tissues andadjacent normal tissues were quantitative mass spectrometry by usingiTRAQ technology, Screening molecular markers of HBV infection relatedHCC, The new discovery of candidate markers and the part that is not in theHCC study reported protein were further verified at gene and proteinexpression by Realtime PCR and Western.2. To verify the differentvalidation expression in HBV positive and negative hepatocellularcarcinoma adjacent tissue by using tissue chip technique for some not yet inthe HCC study reported protein.3.Using HBV replication cell model ofHepG2.2.15cells with siRNA and pcDNA3.0gene eukaryotic expressionplasmid Technology, the selected related to HBV infection and specificmolecular markers of hepatocellular carcinoma in gene knockout andtransfection.To detect the HepG2.2.15cell candidate markers proteinexpression by Western blot analysis, and to preliminarily analysis theeffects of the expression level of gene induction and the candidate symbolcorrelation and HBV replication of type I interferon and5downstreaminterferon.Results:(1)Using iTRAQ combined with mass spectrometry tocomparatively analyze the differences between the9double cancer，tissuesand paracancerous tissues’differences in expression profiles in patients withHCC related to HBV infection, and differential expression of222HCC and the detection of cancer tissue and paracancerous tissues obtained from1608proteins of at least1.3times the protein. Among them, the HSPB1, Villin-1,AKK1B10, APOE, UGDH, CSTB, G6PD, HIST2H2BF, HNRNPA1, GSN,NPM1, S100A6, TKT, VLL1, ADH4, ADH1B, ADH1C, CYP1A2,UGT2B4,the relative mRNA expression in cancer tissues compared withthat in adjacent tissue are up-regulated in HSPB1, Villin-1, AKK1B10,APOE, UGDH, CSTB, G6PD, HIST2H2BF, HNRNPA1, GSN, NPM1,S100A6, TKT, VLL1and ADH4are up-regulated, while in ADH1B,ADH1C, CYP1A2, UGT2B4are down-regulated. Western blot verificationshows: cancer tissues by G6PD, HSP27, APOE, S100A11, Villin-1expression increased significantly compared with the adjacent tissues, andthe expression of ADH1B was significantly down-regulated. Western blotand Realtime PCR at the protein and mRNA levels’ further test and valitedresults are consistent with the screening results of iTRAQ combined withmass spectrometry. Different validation expression followed by HBVpositive and negative for G6PD protein by using tissue chip technique ofhepatocellular carcinoma and normal tissues. Then verify the differentvalidation expression in HBV positive and negative hepatocellularcarcinoma adjacent tissue by using tissue chip technique. Detection of G6PDexpression in HBV positive cancer tissues was significantly higher than thatof HBV negative HCC carcinoma and adjacent tissues, and has nothing to dowith the level of AFP.(2)Using HBV replication cell model of HepG2.2.15 cells with siRNA and pcDNA3.0-G6PD gene eukaryotic expression plasmidwere transfected gene knockout, then analysis and detect candidate markersprotein expression of the HepG2.2.15cells by Western blot. A furtheranalysis on the correlation between G6PD and HBV viral replication and onthe effect on expression level of gene induction of type I IFN and5Idownstream IFN.G6PD is an endogenous factor promoting HBV viralreplication, and may influence the host cell type I interferon and5downstream interferon induction gene expression level.Conclusion:1.iTRAQ technology is a powerful and reliableperformance research tool in screening HCC potential molecularmarker.2.Protein participates in the occurrence and development process ofrelated HCC HBV infection is extremely complex.3.The gene and proteinlevels showed expression of G6PD in HBV positive HCC in cancer tissueswas significantly higher than that in HCC tissue adjacent to cancer, and hasnothing to do with the level of AFP. At the same time, tissue microarrayshowed a clear correlation between HBV infection, which shows that G6PDnot only could be used as a potential new specific biomarkers in diagnosis ofrelated HCC HBV infection, but that G6PD may also be involved in theprocess of HBV replication.4.siRNA and pcDNA3.0-G6PD gene knockoutand transfection confirmed that G6PD is involved in the regulation of HBVreplication, and may be an endogenous promoting factor for HBVreplication.5.G6PD may be involved in the regulation of host cells by the interferon stimulated gene pathways and influence of HBV virus replication,its mechanism still need further study.6.siRNA inhibited the expression ofG6PD gene may be a kind of new drugs against HBV potential biologicaltarget therapy. Objective: Explore the relevance of CHB patient’s age, sex, medicalhistory, serum HBVDNA levels, liver function and serum levels ofPDGF-BB and Liver Fibrosis (HA, C Ⅳ, PC Ⅲ, LN), and relevancebetween serum levels of PDGF-BB and CHB liver fibrosis degrees.Methods:1.The740cases of CHB patients with serum levels ofPDGF-BB and CHB patient’s age, sex, medical history, serum HBVDNAlevels, liver function and serum hepatic fibrosis markers (HA, C Ⅳ, PC Ⅲ,LN) to conduct analysis of relations.2.The740cases of CHB were dividedinto3groups, mild, moderate and severe and analysised the relevance ofserum HBVDNA level, PDGF-BB levels and hepatic fibrosis and liverfunction classification.3.To compare and analysis to liver fibrosis index(HA,CⅣ,PCⅢ,LN) and serum PDGF-BB levels of patients ofHBeAg-negative and HBeAg-positive chronic hepatitis B (CHB).Results:1.Serum levels of PDGF-BB in patients with CHB age, sex,medical history, HBVDNA have not significant correlation (p>0.05); but liver function parameters and serum HA, PC Ⅲ, C Ⅳ, LN was significantlycorrelated (P <0.01).2.PDGF-BB levels in serum and liver fibrosis HA, PCⅢ, C Ⅳ, LN level and liver function was positively correlated (P<0.01).3.PDGF-BB levels in serum hepatic fibrosis HA, PC Ⅲ, C Ⅳ, LNlevel was positively correlated (P <0.01).4.serum HA, PC Ⅲ, LN, C Ⅳ andserum PDGF-BB levels of HBeAg-negative group was higher thanHBeAg-positive group, there was significant difference (P<0.05).Conclusions:1.The extent of the development of liver fibrosis in CHBis not determined by age, sex, the length of a history and the level of HBVDNA, liver inflammation activity is the beginning promote in thedevelopment of liver fibrosis,which decide the extent of fibrosis; the levelsof serum PDGF-BB is not only can reflect the CHB patients with liverdamage but also can reflect the degree of liver fibrosis.2.It can be used inCHB liver function and liver fibrosis assessment.3.Serum PDGF-BB levelsof HBeAg-negative CHB be higher than HBeAg-positive CHB may be oneof the reasons of HBeAg-negative CHB liver fiber the degree be severer thanthe HBeAg-positive CHB. |
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