Expression On Bim In Apoptosis Of PBMC Of In Chronic Hepatitis B Patients

Objective:Chronic hepatitis B is one of infective diseases to threaten human health which is mainly caused by continuing exist of hepatitis B virus. Hepatitis B virus damages hepatocytes by activating immune system. Repeating inflammations in the liver will lead to hepatic cirrhosis, even hepatic carcinoma if virus cannot be completely cleared.Immune response of host determines the generation,development and outcome of diseases after hepatitis B virus infects human body. CD4+T cell and HBV-specific CD8+T cell in chronic hepatitis B patients decreased,so that immune function of this patients was low and unable to control HBV infection. However,the hypocellular mechanism was not clear. Recently peripheral blood lymphocytes which were stimulated by virus,bacterial infection or mitogen began to activate and proliferate.During the shut-down of the immune response activated lymphocytes were removed by apoptosis. This phenomenon was called activation induced cell death(AICD). Many researches have found that the ratio of apoptosis in chronic hepatitis B patients was higher than normal control which indicated that HBV was able to induce more lymphocytes to apoptosis so that immunocytes decreased and could not clear HBV. Excessive apoptosis in AICD may be one of mechanisms which resulted in chronic hepatitis. Many studies have found that the death receptors such as FasL and TRAIL of peripheral blood mononuclear cells in chronic hepatitis B patients expressed more than normal control which may be the cause of apoptosis. Intrinsic pathway is another way leading to cell apoptosis which is mainly regulated by Bcl-2 family. Whether it is involved in AICD is uncertain. Bim is one member of Bcl-2 family which contain one BH3 domain. It plays an important role in maintaining both center tolerance and peripheral tolerance. Some researches have found that apoptosis regulators Fas and Bim cooperated in shutdown of chronic immune responses and prevention of autoimmunity. So we mimicd the process of AICD in vitro to explore other mechanism to provide a theoretical basis on the treatment of chronic hepatitis through this experiment. we studied the effect of Bim in apoptosis of peripheral blood mononuclear cells in chronic hepatitis B patients and normal control. At the same time we investigated the correlation between apotosis and Bim. We further explored the mechanism of apoptosis in PBMC in chronic hepatitis B patients.Methods:1 Subjects of research: Thirty patients with chronic hepatitis B (sixteen men and fourteen women,age from eighteen to fifty-five ,Mean age thirty years) were enrolled. All subjects were diagnosed according to the guide of preventi- on in chronic hepatitis B which was established in 2005 by Chinese Medical Association. All subjects showed no evidence of alcoholic,autoimmune or dr- ug-induced liver disease. None have undergone antiviral or immunomodulato- ry therapy. We have excluded other virus infections such as HAV,HCV,H- DV and HEV and acute infection. Health donors fifteen(nine men and six wo- men,age from eighteen to fifty,mean age twenty-nine years) were enrolled.2 Measure the content of HBVDNA through RT-PCR.3 PBMCs collection and cells culturePBMCs were extracted from fresh and heparinezed blood by through a Ficoll-Hypaque density gradient centrifugation in an asepsis condition. PBMCs were stimulated by PHA for 16～18h , then removed PHA from nutrient medium and added exogenous IL-2 . Cells were cultured at least 7 days and stimulated again by anti-CD3 antibody for 48 hours.4 Detection of Cell apoptosisPBMCS were fixed by 70% ethanol at 4°C overnight. Washed twice with PBS(phosphate buffered saline),Cells were incubuted with about 1000μl of solution(include Rnase A PI) in room temperature about 30 min and analyzed using flow cytometer.5 Detection of Bim proteinProteins were extracted from already dealed cells and preserved under -80℃. We collected enough samples and detected the expression of Bim by Western blot.Result:1 The ratio of apoptosis in PBMC of chronic hepatitis B patients was higher t- han normal control. The discrepancy was significant ( t=3.592,P<0.01). The ratio of apoptosis in PBMC of patients with HBVDNA positive was higher t- han HBVDNA negative. The discrepancy is significant(t=2.745,P<0.05).2 The expression of Bim in PBMC of chronic hepatitis B patients was higher than normal control. The discrepancy was significant (t=4.588,P<0.01)). The expression of Bim in PBMC of patients with HBVDNA positive was higher than HBVDNA negative. The discrepancy was significan(tt=3.262,P<0.01).3 The expression of Bim had positive correlation with apoptosis.(r=0.750,P<0.01)Conclusion:1 The ratio of apoptosis in PBMC of patients with chronic hepatitis B was hig- her than normal control which may be one important cause to lower immune function of chronic hepatitis B patients.2 The expression of Bim in PBMC of patients with chronic hepatitis B was higher than normal control. It had positive correlation with apoptosis. The result illustrated Bim was involved in AICD and may be an important mechanism to lower the number of T cell in patients with chronic hepatitis b.