Study On Immunoregulation And Telomere System Of Aplastic Anemia Treated With Androgen

Objective To investigate the role of CD4⁺CD25^highCD127^low regulatory T cells in the pathophysiology of AA and the effect of androgen to regulatory T cells in AA patients.To further explore the effect of androgen on telomeres and telomerase system in AA patients,and the role of telomerase mutation,telomere length and telomerase activity in the pathogenesis of AA.Methods The patients were collected according to the AA clinical diagnostic criteria,and were treated according to the patient's individual condition,the diagnosis and treatment standard.?ATG+CsA,CsA+danazol and danazol respectively.The peripheral blood samples were counted by multi-color flow cytometry,and the percentage of T-regs/CD4⁺T lymphocytes was calculated.The results were compared with healthy control group.?CsA+stanozolol and CsA respectively.The genomic DNA of peripheral blood was extracted by nucleic acid extractor and nucleic acid extraction kit.The relative length of telomere in peripheral blood leukocytes was measured by real-time fluorescence quantitative PCR.Telomerase activity was detected by ELISA.PCR amplification of TERT and TERC gene,purification,sequencing,analysis,and compared with the healthy control group.Results First Section There was a significant difference between the mean T-regs in the 25 patients?3.45±1.33%?and the controls?7.90±0.99%??p<0.0001?.All patients showed a significant rise in mean T-regs after 3 months of treatment?6.70±1.99%??p<0.0001?.Thirteen patients showed no response,11 patients had partial response,and 1patient had a complete response.The mean rise in T-regs was higher in the Responders group than the Non-responders group?p=0.613?.The mean rise in T-regs after 3 months of treatment was not significantly different among three groups?p=0.585?,but did increase significantly in all three groups?p<0.0001?.No significant predictors of change in T-regs'value were found by univariate analysis.Second Section There were 1 TERC1 mutations in the 30 patients,and no mutations of TERT1,TERT2 and TERC 1 were found in healthy controls.There were multiple Single Nucleotide Polymorphism loci of TERT and TERC genes in both the experimental group and the healthy control group.The relative T/S ratio of the baseline telomere length in AA patients was 0.765±0.271,which was significantly shorter than that in the normal control group?0.978±0.344??P=0.028?.12 months after treatment,the relative T/S ratio of the telomere length in the CsA+androgen group was higher than that in the CsA group?P=0.017?.The mean value of baseline telomerase activity?OD value?in the experimental group was 0.30±0.16,which was significantly higher than that in the normal control group?0.11±0.06?.In CsA+androgen group,telomerase activity?OD value?decreased 3 to 6 months after treatment,but increased at 9-12 months.There wasn't significant difference between groups.After treated with CsA,3?20%?and 6?40%?patients acquired hematological response in 3 and 6 months,but no new hematological response was found until 12months.After treated with CsA+androgen,8?53.3%?,10?66.7%?,11?73.3%?and12?80%?patients acquired hematological response in 3 to months.The improvement rate of hematological indexes in the CsA+androgen group were better than that in the CsA group?P=0.03?.There were no grade 3 or grade 4 adverse drug events in all the patients.One patient had"liver benign space occupying lesion".Mild renal function injury occurred in one case.The mild damage of liver function was observed in 2cases of that patients.Conclusion First Section T-regs play an important role in the pathophysiological process of AA.Androgen may have immunoregulation effect,but the mechanism of androgen on AA may be mainly due to its effect on telomere,which is the direction we need to further study.In general,danazol as a weakened androgen may have immunoregulation potential.Second Section Androgen can regulate telomerase activity and telomere length in AA patients,and the increase of telomere length after androgen therapy is consistent with the regulation of telomerase activity by androgen.Androgen is effective in the treatment of AA with telomere dysfunction and clinical hematologic improvement can be achieved.There are no serious side effects of androgen in AA treatment.