| Backgroud and ObjectiveLiver cirrhosis and hepatic carcinoma related to hepatitis B virus(HBV)infection are still the main problem threatening the human healthy in the world.Early diagnosis,early findings and immediate measurements in the population with high risk of HBV infection are very important to improve the prognosis of liver cirrhosis and hepatic cellular carcinoma.The purpose of this study is to analyze the correlation of HBV DNA mutation and change of some cytokine levels with liver cirrhosis and hepatic carcinoma,in order to find out novel pathological molecular targets,hence providing clinical and basical evidence for further specific treatmrnt of HBV induced chronic liver diseases.Methods1.Clinical study:127 cases of chronic hepatitis B(CHB),65 of liver cirrhosis and 45 of hepatic carcinoma with confirmed HBV infection were enrolled in this study.DNA sequencing,mismatch amplifying mutating PCR were used for detection of the mutation at the sites of HBV basic core promoter(BCP)T1762/A1764 and precore A1896,PCR-fluorescent-probing assay for HBV DNA levels,enzyme-link immune adsorbed assay for serum cytokine levels.Immuno-histochemistry assay(IHC)for location of cytokine expression in the tissue of liver carcinoma and around parts,realtime PCR for gene expression levels in cells or tissues.2.In vitro cell study:CCL5 siRNA was transfected into hepatic carcinoma cell line HepG2,scarification test and transwell assay were performed to observe cell migration activity and capacity,Western blotting analysis for protein expression.Results1.Study on the relationship between HBV pre-core,BCP mutation and chronic liver diseases:(1)Incidences of mutation for A1762T,G1764T in BCP and G1896A in precore were 62.48%(148/237),62.48%(148/237)and 49.79%(118/237),respectively,and higher than in other sites,moreover,A1762T and G1764A mutaions often occurred simultaneously;(2)A1762T/G1764A and A1762T/G1764A/G1896A mutation promoted HBeAg conversion,increased serum AFP expression,and were closely associated with the development of liver cirrhosis and hepatic carcinoma;(3)A1762T/G1764A mutation was positively correlated with hepatic carcinoma incidence;(4)G1896A increased HBV DNA replication.2.Study on the correlation of serum cytokines and HBV-associated liver cirrhosis/hepatic carcinoma:(1)serum HA,LN,PC-III and C-IV levels were lower in chronic hepatitis B patients than in patients with decompensated liver cirrhosis(P<0.001);(2)ROC curves for precisely distinction of liver cirrhosis and CHB showed that AUC value for CCL5,MIP-lb and HA were 0.8011、0.706 and 0.752,respectively with All AUC values bigger than 0.7.CCL5,MIP-lb and HA,therefore,were effective biomarkers for accurately distinction between liver cirrhosis and CHB,among them,CCL5 was better than HA,then than MIP-1b.3.Study on role of CCL5 and its receptors in hepatic cellular carcinoma developments:(1)CCL5 and CCR5 were extensively expressed in carcinoma tissues,obviously higher than in liver cirrhosis tissues;(2)CCR5 specific siRNA significantly downregulated the CCR5 transcription in HepG2 cells;(3)with CCR5 downregulation,24 hours scarification test showed that 50%of scarification was repaired by cell migration in control group,while only 23%was repaired in CCR5 siRNA treated group;(4)In transwell experiment,the migrating cell numbers were 68.73±4.024 in control group,but the numbers were reduced into 24.53±2.545 in CCR5siRNA group(P<0.001).Conclusions1.Mutations on the sites of HBV precore and BCP altered viral replication ability and were closely associated with liver cirrhosis and hepatic cellular carcinoma developments.2.CCL5,MIP-1b and HA levels,especially CCL5,were considered as the parameters for judging the severity of liver cirrhosis and chronic hepatitis B.3.The expressions of CCL5 and its receptor were increased in hepatic cellular carcinoma tissues,it was suggested that they play an important role in the process of migration and evasion of hepatic cellular carcinoma. |
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