Study On TGF-β₁ And N-Glycome In Liver Fibrosis And HCC With HBV Infection

Chronic hepatitis B(CHB) is one of the most common diseases and patients with CHB are at a higher risk of developing hepatocellular carcinoma(HCC).About half a million Chinese die from HCC caused by HBV and from end stage cirrhosis each year.The patients with chronic HBV are at increased risk of progression to cirrhosis and development of HCC.Liver cirrhosis in its early stage is able to reverse,so it is important for diagnosis and treatment of liver diseases to early diagnose of liver fibrosis. Unfortunately,it is still a challenge in clinical practice.The project is divided into three partsPartⅠ:Influence of anti-fibrosis cytokine on activities of transforming growth factor-betal gene promoter.To study the effect of anti-fibrosis cytokine(IL-10,HGF,IFN-γ) on the activities of transforming growth factor-betal gene promoter containing -509C＞T.The recombinant plasmid phTGF2.14C/phTGF2.14T containing 5'flank sequence of transforming growth factor-betal gene and chioramphenicol acetyltransferase(CAT) was transfected into Zhang's hepatocyte.The activities of CAT were assessed by ELISA method 24h after cytokine treating.(1) In control group:the activities of CAT in Zhang's hepatocyte after transfection of phTGF2.14C were significantly higher than transfected by phTGF2.14T. (t=12.5882,P=0.0002).(2) IFN-γhad marked inhibition effect on the activities of transforming growth factor-betal gene promoter including -509C＞T respectively. (phTGF2.14C and phTGF2.14T vs.control group,t=11.9183,P=0.0003,t=4.6154,P= 0.0099).In promoter sequence of transforming growth factor-betal containing -509C＞T, allele C had obviously enhanced activities of promoter.As the anti-fibrosis cytokine, IFN-γhad marked inhibition effects on the activities of transforming growth factor-betal gene promoter,however,cytokine(IL-10,HGF) had no obviously effect on sequence 2.14Kb(-1328～+812bp),no matter promoter containing -509C or -509T.PartⅡ:Profiles of N-glycome in sera of patients with liver diseasesTo compare the expression profiles of serum glycomics between liver fibrosis hepatocellular carcinoma(HCC) patients and normal adults,in an attempt to search for new biomarkers for liver diseases.Serum samples were obtained from liver fibrosis(n=69) HCC(n=100) patients with hepatitis B virus(HBV) infection and normal adults(n=130). Profiles of serum N-glycome were determined by capillary electrophoresis(CE) based on DNA sequencing equipment.Each sample had a profile with 10 peaks;the peaks were adjusted and quantified in form of relative percentage.Peaksl,4 in liver fibrosis group were higher than those in the control group;Peaks 5,7 and 8 were lower in liver fibrosis than those in control group(p＜0.05).Peaks 1,2,9 and 11 in HCC group were higher than those in the control group;Peaks 6,8 and 10 were lower in HCC group than those in control group(p＜0.05).And the changes of peak values of several peaks were correlated with routine clinical laboratory parameters,such as alpha-fetoprotein(AFP),HBV-DNA copies,albumin and prothrombin time.N-glycome profiles in serum are significantly different between liver diseases and normal adults which may provide evidence for clinical diagnosis of liver diseases.PartⅢ:Analysis of the clinical laboratory parameters on hepatocellular carcinoma (HCC)To search parameters related to the diagnosis of hepatocellular carcinoma by analyzing the clinical laboratory indexes in HCC.Collect clinical laboratory data of 828 patients who were diagnosed as HCC before operation,such as tumor makers,serological biochemical indexes,hepatitis B virus(HBV) infection markers,HBV DNA titers,and then analyze their correlation with tumor size and the pathological grades of HCC.It was found that,97.9%of the 828 cases were infected with HBV,and 70.9%were accompanied by hepatic fibrosis.Among the HCC patients,tumor size was related to ALB,GLB,A/G,AST,AST/ALT,GGT,ALP,AFU,AFP and tumor grades;meanwhile the pathological grades of tumor,were related to PALB,GGT and tumor size(P＜0.05).The progression of HCC was correlated with several clinical lab biochemistry indexes and viral makers.So the combined application of these indexes is worth to be further studied for auxiliary diagnosis of HCC.Based on the pathogenesis mechanism of liver disease,this study aimed to find the relation between cytokines and liver disease on the gene level so as to provide solid base for the following clinical research.Apply new N-glycome analysis technique into liver disease research,find the representative glycome of liver diseases,and then provide a technique for its diagnosis.Meanwhile selection the closely related indexes in common clinical laboratory examination is in the hope to establish an effective multi-parameter diagnosis model for HCC.