The PD-1Demethylation Pattern In Its Promoter Region Of CHB Patients

Part one:The expression and influence factors of programmed death receptor1on peripheral blood mononuclear cells of chronic hepatitis B virus-infected patientsObjective To observe the expression variance of Programmed death one (PD-1) on PBMCs and CD4+T/CD8+T lymphocytes in Chronic Hepatitis B patients and further investigate The relationship between the probable influence factors and the PD-1expression on peripheral blood mononuclear cells (PBMC) in CHB Patients.Methods A total of116subjects, including63patients with chronic hepatitis B(CHB),35healthy hepatitis b virus carriers,18healthy blood donators were enrolled. The expression of PD-1, CD4and CD8on the peripheral blood mono-nuclear cells (PBMCs) were detected by FCM; The serum HBV markers, HBV DNA load and liver function were also measured. The difference of PD-1expression on PBMCs, CD4+Tand CD8+T lymphocytes were analyzed in chronic hepatitis B and hepatitis B virus healthy carriers and healthy people enrolled in this study. The relationship between the HBV-DNA, ALT level and hepatitis B e antigen were further observed in CHB patients.Results Taken the PD-1expression in normal controls as a baseline level, the expression of PD-1on PBMCs and CD4+TT lymphocytes in CHB patients and hepatitis B virus healthy carriers was significantly increased; hepatitis B virus healthy carriers was much higher than CHB patients. PD-1expression on CD8+T lymphocytes had significantly difference only between hepatitis B virus healthy carriers and normal controls, but no significant difference between other groups. In CHB patients, the PD-1expression in PBMCs from patients with HBeAg positive in serum was much higher than that from those with HBeAg negative in serum, And the PD-1expression level not correlated with serum HBV DNA load and serum ALT level.Conclusion The PD-1expression level on CHB patients is much higher than normal controls but significantly lower than hepatitis B virus healthy carriers. PD-1expression on peripheral blood mononuclear cells (PBMC) may Associated with the immune state in chronic HBV infectors. Long-term exposure to HBV e antigens in CHB patients may impact the PD-1expression level on PBMCs. Part two:Effects of5-Zac on demethylation pattern of the PD-1gene in promoter region and PD-1expression in a human T lymphocyte cell lineObjective To observe the DNA demethylation of the PD-1promoter caused by5-azacytidine (5-azac) in Molt-4cells (T lymphocyte cell line) and further investigate the relationship between the expression of PD-1and DNA demethylation.Methods The Molt-4cells were cultured in medium containing different concentrations of5-azac(0,5,10Umol/L)for72h;The expression of PD-1in Molt-4cells and the apoptosis were detected by FCM; the mRNA transcription level of PD-1was detected by RT-PCR;Molt-4cell DNA in all groups were treated by sodium bisulfite; The PD-1promoter fragment was amplified by PCR,these amplification fragments were transformed into E. coli. Positive clones were selected for equencing, methylation status of the fragments of PD-1promoter was examined.Results5-azac(1.13%±0.01%) treated cells was found more lower than that in both5μmol/L and10μmol/L5-azac treated cells (18.96%±1.87%,63.09%±6.25%,P<0.05), and they showed concentration-dependent (P<0.01). Cells apoptosis rate and PD-1mRNA expression were also observed increased significantly with5-azac treating. The results of bisulfite genomic sequencing showed that Demethylation probability of CG points on-601bp and-553bp were significantly increased in the5-Zac treated cells compared with the untreated cells.Conclusion5-azac inhibits cell grouth in human lymphoid cell series Molt-4by inducing PD-1gene expression and promoter demethylation. PD-1gene promoter demethylation may be one of the important mechanisms for PD-1increased expression in5-azac treated Molt-4cells. Part three:The methylation pattern of PD-1promoter region and its influence factors in peripheral blood mononuclear cells of chronic hepatitis B virus-infected patientsObjective To observe the relationship between the expression of the Programmed death one (PD-1) and DNA demethylation pattern of the PD-1promoter on PBMCs in Chronic Hepatitis B patients and further investigate the effect of hepatitis B virus e antigen on methylation pattern of PD-1promoter region in peripheral blood mononuclear cells (PBMC) of CHB Patients.Methods20patients with chronic hepatitis B(CHB), including10HBeAg positive and10HBeAg negative,18healthy blood donators as control were enrolled. The present study detected the correlation between the PD-1mRNA level in the peripheral blood mononuclear cells of the chronic hepatitis B patients and serum HBeAg levels, then determined the methylation of the promoter region of the PD-1gene using sodium bisulfite sequencing, so as to investigate the effect of HBeAg on PD-1expression and methylation of promoter region in peripheral blood mononuclear cells of chronic hepatitis B patients, and the relationship between them.Results the PD-1mRNA level of the peripheral blood mononuclear cells in the normal control group was (0.10±0.023), which was significantly lower than those in the HBeAg (+) group (0.44±0.046)and the HBeAg (-) group (0.18±0.014)(P<0.05), and the expression rate was higher in the HBeAg (+) group than that in the HBeAg (-) group (P0.05). the correlation analyses between the PD-1expression and the probability of methylation in each locus showed that the probabilities of methylation at-601,-553,-538,-483,-463and-317were correlated with the PD-1expression (P<0.05), Further comparison of the difference of the methylation status of the methylated loci in the promoter of the PD-1gene in the normal control, HBeAg (-) and HBeAg (+) groups demonstrated that the methylation levels at the CpG loci of-553bp,-538and-483were gradually declined in the normal control, HBeAg (-) and HBeAg (+) groups, and significant difference was observed among groups (P<0.05).Conclusion the methylation state of some CpG loci in the promoter region of the PD-1gene is associated with the PD-1expression, and the effect of HBeAg on the PD-1expression in the peripheral blood mononuclear cells of chronic hepatitis B patients may be related to the impact on the demethylation of the CpG loci in some binding sites of the promoter and transcription factor of PD-1.