The Study On Fetuin A And Its Fucosylated Glycosylation In HBV Related Liver Diseases

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related death in the word, with the high death, high recurrence, poor prognosis characteristics.The5years’survival rate is less than10%. Hepatitis B and C is the risk fators for the global HCC disease.The highest incidence of the world’s hepatocellular carcinoma occurs in china. Serum protein AFP and ultrasound examination are now used for HCC high-risk screening, but both of them have defects of low sensitivity. Most patients has reached at an advanced stage or transferring when diagnosed ash liver cancer It is important to seek high sensitivity and specific new liver cancer markers refers to the high mortality rate.In recent years, with the progress of the study of glysosylation, abnormal glysosylation in liver cancer research has made considerable progress, and provides a new basis for early diagnosis markers and pathogenesis research. Callewaert etc have got HCC serum total protein N-glycan profiling with DSA-FACE method, found that some typical glysosylated structures appear in some serum proteins, and could be used to monitor the liver fibrosis,cirrhosis, and liver tumors development.The change of fucosylation is particularly remarkable in all kinds of abnormal glycosylated modification. Fucosylated protein (AFP-L3) in2005was adopted by the FDA as liver cancer diagnosis and prognosis index, AFP-L3level was related to liver cancer stages and liver cancer cell biological behaviours. Comunale etc identified about20different fucosylated proteins between liver cancer group and the normal group, including immunoglobulin molecules and the other serum glycoproteins.The level of fucosylation is related to the biological actions of liver cancer cells, and the occurrence\prognosis and development of liver cancer.These glycoproteins are considered as promising liver cancer markers.Serum protein Fetuin A is secreted into the blood by liver. It is a natural insulin receptor inhibitors and systemic ectopic calcification inhibitors, and is related to insulin resistance and liver fat accumulation. Liver cancer patients have insulin resistance, glucose metabolic disorder and calcium phosphate metabolic disorders, its mechanism is as likely to involve with Fetuin A molecules.The reduced level of serum Fetuin A was shows as a short-term independent predictor of death for liver cancer patients, and was related to liver cell function decompensated. Fetuin A combined with the tumor cell surface receptor via calcium ions, to promote tumor cell growth, inhibit the liver inflammation occurrence and reduce the liver cell apoptosis. Fetuin A may also play an important role in transferriong and growth of cancer cells in the blood vessels.This research was to discuss specific expression and diagnostic value of serum protein Fetuin A in primary liver cancer, liver cirrhosis with large sample of clinical case.Part1:Fetuin A expression levels in serum of HBV related liver diseases and tissues of the patients with hepatocellular carcinoma and its clinical significanceThis study aims to assess diagnostic value and application of the serum Fetuin A level in primary hepatocellular carcinoma (HCC), cirrhosis, and the healthy control group.We collected144cases of liver cancer, liver cirrhosis, and the healthy control.The levels of serum Fetuin A protein of each group were detected by ELISA system.We found that the serum level of Fetuin A protein in liver cirrhosis group was significantly lower than the liver cancer and health control group (p<0.001, respectively). The difference between the liver cancer and healthy control group was not statistically significant.Fetuin A serum protein level was negatively correlated with laboratory indexes such as TBA, PT, APTT, PLT, IBIL TBIL, DBIL as well as the Child-Pugh grade;and was positive correlated with the laboratory indicators such as RBC, TP, ALB, A/G, AFU, CA199and HGB.To the detect the tissue Fetuin A protein level in patients of liver cancer, We Collected26cases of hepatocellular carcinoma patient,and get26pairs of cancer tissues and the adjacent tissue parts. The levels of Fetuin A protein of each pair were detected by Western-blot. We found that Fetuin A protein expression levels in liver cancer tissues was significantly higher than the corresponding adjacent normal tissues (P=0.002).Part2:Establishment of Lectin-ELISA method to detect the fucosylated level of serum Fetuin A.This study created a Lectin-ELISA method to detect the fucosylated level of serum Fetuin A.This method is similar to the antibody sandwich ELISA,the glycoprotein structure-specific binding lectin was used instead of second antibody.Fetuin A antibody was coated on ELISA plate to grab the sample antigen.Fucose-specific lectins recognize the fucosylated glycosylation structure of antigens.It was used to detect the fucosylated level of antigen. Optimization of working concentrations of coated antibody\lectin\sample dilution was determined.Since there is no commercial fucose glycosylated Fetuin A standard, mixed serum was used as standard to product standard curve.Limit of detection, cross-reactivity, interference experiments with serum bilirubin were used to evaluate the ELISA method. The Fetuin-A antibody and lectin AAL optimal working concentration were lug/ml and0.4ug/ml, and optimal serum dilution was1:100. The intra-and inter-assay variations were6.91%and12.2%, respectively. The minimum detection limit was1.95ng/ml. There is no cross-reactivity with homologous sequences cystain C and human IgG. The free bilirubin in serum has interference effects on the absorbance value of the method.Part3:The expression levels of fucosylated glycosylation of Fetuin A in serum of HBV related liver disease and its diagnostic applications.The purpose of this study is to analyze the expression levels of fucosylated glycosylation of Fetuin A in the serum of patients with liver cancer, cirrhosis, HBV asymptomatic carriers and healthy controls; and to investigate the diagnostic value of fucosylated Fetuin A. Lectin-ELISA method was used to detect the serum fucosylated Fetuin A level.We collected144cases of HCC, liver cirrhosis and health controls. We found that serum fucosylated Fetuin A level in HCC and liver cirrhosis was significantly higher than that in healthy control group. And the fucoslated ratio of Fetuin A in serum (serum fucosylated Fetuin A level/serum Fetuin A level) was significantly higher in HCC and liver cirrhosis than that in healthy control group.Then we collect122cases of HCC,61patients with liver cirrhosis,116HBV asymptomatic carriers and108healthy controls to validate the once obtained results. The diagnostic value of Fetuin A fucosylated level in liver cirrhosis was analysed with receiver operating characteristiccurve (ROC).The study found that:The fucosylated level of serum Fetuin A in HCC and cirrhosis group were significantly higher than that of asymptomatic carriers of HBV and the healthy control group, the difference was statistically significant (P<0.05).The difference between the Asymptomatic carriers of HBV and healthy control group was not statistically significant. Using Cutoff value of1.12to identify cirrhotic patients from asymptomatic carriers of HBV and the healthy control group, the area under the ROC curve was0.906, the95%confidence interval was0.861to0.951. The fucosylated level of serumFetuin A was negatively correlated with laboratory indexes such as ALB, PALB, TP, PLT, UA, RBC and HGB,and was positively correlated with laboratory indexes such as ALT, AST, ALP, ADA, of AFP, CEA, CA199, CA125, DBIL TBIL, LDH, GGT, TBA and PT.