| Backgroud:Hepatitis B is among the most common infectious diseases and has variable prevalence in the world. There are about 350 million people with chronic hepatitis B(CHB), a potentially progressive fatal disease. According to the report of the World Health Organization, hepatitis B has become the ninth cause of human death. In China ,hepatitis B virus carriers account for almost 10% of the total population,that is, 120 million people. About 15% ~ 40% have developed into such different types of liver disease as active hepatitis, cirrhosis and even hepatocellular carcinoma, and the incidence of hepatitis B in China is increasing.The outcome of hepatitis B virus (HBV) infection is decided by the interplay between the virus, of which genetic mutations have been considered one of the basic factors influencing HBV biological features, hepatocytes, and the host's immune response. Certain HBV mutants may change the ability of replication and pathogenicity of the virus,alter HBV antigen epitope which is related to either an exaggerated host immune response and is likely to cause severe damage of hepatocytes. Research shows that mutations in precore(PreC)/ basic core promoter(BCP) can abrogate HBeAg expression/secretion. It is suggested that secretary HBeAg will alleviate injury of HBcAg-expressing hepatocytes by cytotoxic T cells (CTL), for HBeAg and HBcAg share CTL immunological epitopes. Therefore, precore/BCP mutations may have negative effect on immunotolerance but have something to do with the aggravation of chronic hepatitis B. However, researches have different opinions on the above-mentioned opinions.HLA-â… (Human Leukocyte Antigen classâ… ) is encoded by the human major histocompatibility complex and widely exists in the nucleated cell surface. HLA-â… antigen plays a key role in the immune system because it is one of the main elements that participate in specific immune recognition and immune response. HLA-â… in membrane surface of the normal liver parenchyma cells expresses little hardly expresses but expression is unusual after HBV infection. Serum soluble HLA-â… (sHLA-â… ) and membrane surface HLA-â… have the same polymorphic determinant specific. sHLA-â… is not only very low but also relatively constant in the normal person serum,but its level increases remarkably after HBV infection. This article is to study the association of HBV PreC/ BCP mutations and the level of serum sHLA-â… with the progression of liver disease, and to study the correlation between them.Research subjects: The patients with chronic HBV who receive treatment in Fenyang hospital are subjects in the experiment.Objectives:(1)To compare the influence of three different methods in extracting HBV DNA on PCR products.(2)To discuss the relationship between the HBV PreC/BCP mutation and the level of the serum sHLA-â… ,and the occurring,developing,prognosis of the hepatitis B. And to study the effect of HBV mutation on sHLA-â… level from the perspective of immunity.Methods:(1)Serum samples of 133 patients with HBsAg carriers and 20 healthy humans were collected from Shanxi Medical University Fenyang Hospital.(2) Treatment of samples: 3 methods i.e. alkaline lysis ,boiling,boiling lysis were used for extraction of serum HBV DNA. The obtaining rate and optical density value of DNA stripes of DNA stripes are compared to look for a suitable template extraction method.(3) HBV genes were amplified by nested PCR. To design two different sets of nested primers which were located at PreC gene,BCP gene respectively.(4)PCR product were analyzed by restriction fragment length polymorphisms(RFLP)to determine the HBV mutant, wild-type and mixed type of infection. To confirm the accuracy of this method a number of products were picked randomly to measure its sequencing.(5) The serum levels of sHLA-â… in healthy bodies and HBV patients were detected by ELISA (enzyme linked immuno-sorbent assay).(6)Statistics was done with SPSS13.0.Results:(1) The treatment of samples with alkaline lysis for the detection of HBV DAN is more sensitive than the other methods(P<0.01).(2) The double mutation in BCP(T1762/A1764) was obviously more frequent in CHB (58.2%)than that in precoreA1896 mutation(15.2%,P<0.001).(3)There is remarkable correlation between the expression of HBeAg and double mutation of BCP(T1762/A1764)(P<0.01),but no obvious correlation exists between the expression of HBeAg and precore(A1896) mutation(P>0.05). There is a significant relationship between precore(A1896)/ BCP double mutant and the disease progress in chronic hepatitis(P<0.05;P<0.01), but there is no significant difference among mild, moderate chronic hepatitis and cirrhosis groups(P>0.05). (4)Serum level of sHLA-â… of HBV infections group is obviously higer than that of normal group(P<0.01),the level increases with the severity of the chronic hepatitis(P>0.05 ), and in asymptomatic carriers is also higer than that in normal control group(P>0.05 ).(5) The levels of sHLA-â… is relevant to precore/BCP mutations.The expression of sHLA-â… in mutation groups increases remarkably(P<0.05).(6) The levels of sHLA-â… in groups of HBeAg(+)and HBeAg(-) have no significant difference between them(P>0.05).Conclusion:(1)The alkaline lysis method is the best choice of template preparation.(2)Patients with chronic hepatitis B are more prone to BCP double mutations, and the mutations in HBV precore /BCP may be contribute to hepatitis B severity. The mutation of BCP may serve as an indicator for follow-up of asymptomatic carriers.(3)PreC / BCP mutations affect the patient's immune function.The damage of hepatocyte contribute to the release of sHLA-â… .The level of sHLA-â… is related to the development of hepatitis B. |
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