| Aplastic anemia is a group of chemical, physical and biological factors, drugs and unknown causes of bone marrow stem cells and hematopoietic microenvironment of injury, that bone marrow failure, pancytopenia in peripheral blood of a disease. The etiology and pathogenesis have not yet completely clear, it is more difficult to treat, children with refractory hematological diseases was common. Pathogenesis of aplastic anemia is more complicated.In the past, the incidence of aplastic anemia involved hematopoietic stem cell damage, hematopoietic microenvironment and immune defects. These three mechanisms of individuals in different patients alone or in combination, leading to hematopoietic failure. More and more clinical and experimental study confirmed that immune system disorders including the number of T lymphocytes and hematopoietic dysfunction.These played an important role in the the occurrence and development of the AA. Clinical data showed that about 70% of aplastic anemia patients with immunosuppressive agents such as ATG/ALG and CsA treatment were effective, suggesting that the immune-mediated hematopoietic suppression play an important role in the pathogenesis of AA.Recently, CD4+CD25+ regulatory T cells (Treg) caused widespread concern of scholars home and abroad, it is in the maintenance of peripheral tolerance plays an important role in the clinical findings, CD4+CD25+ Treg cells related to a variety of autoimmune diseases, transplant rejection, Cancer and viral infection. Recent studies have shown CD4+CD25+ T lymphocytes were divided into three phenotypes: CD25high, CD25intermediate and CD25low. Immunosuppressive function of Tregs with high expression of IL-2Ra (CD25), and also expressed transcription factor Foxp3, the phenotype displayed as CD4+CD25high.Recently,some scholars analysis the expression frequency of CD4+CD25+ Treg cells in healthy Chinese, using a three-color immunofluorescence staining, sorting the features with the expression of CD4+CD25int/highCD127low cell populations. Foxp3 by intracellular staining confirmed:CD25int/highCD127low express Foxp3 positive cells, while almost all cells CD25lowCD127high express Foxp3 negative cells. Which prompted CD25int/highCD127low can be used as an characteristic surface markers of Treg cells and sorting for Treg.Transcription factor Foxp3 and nuclear factor of activated T (NFAT) play an important role in Treg development and classification. There experiments confirmed: Foxp3 determine the inhibitory function of regulatory T cells, and these functions may be changed through the translation level of Foxp3. Nuclear factor of activated T (NFAT) is a critical transcription factors of T cell receptor (TCR) mediated signal transduction pathway. NFAT family, including NFAT1,NFAT2,NFAT3 and NFAT4 four members. Solomou such that:The expression of Treg, Foxp3 at the protein level and mRNA level were significantly reduced in the peripheral blood mononuclear cells of patients with aplastic anemia, accompanied with the reduction of NFAT1 protein, and the reduce of NFAT1 protein expression may explain the low expression of Foxp3 and the low frequency of Treg. The current domestic on Foxp3, NFAT1 protein in the pathogenesis of aplastic anemia in children is still rarely reported.ObjectiveIn this study, flow cytometry, Western blot test (Western-Blotting) were used to detect the expression of Treg cells and the transcription factor Foxp3 and NFAT1 in peripheral blood mononuclear cells of children with chronic aplastic anemia(CAA) and normal ones.To explore the relationship between them and immunology in the pathogenesis of aplastic anemia, to provide ideas to the prevention and treatment of aplastic anemia, and to study the immunosuppressive treatment,and may provide the theoretical basis to this treatment.Materials and methods1 MaterialsChoose the children with chronic aplastic anemia among October 2009 to June 2010 in the pediatric inpatient and outpatient of The Third and The First Affiliated Hospital of Zhengzhou University, be divided into two groups:initial treatment period 22 cases and recovery period 22 cases, each group 12 males and 10 females, aged from 2 to 11 years old; normal control group without no recent infection and immune-related diseases in surgery of our hospital, the total number was 15 patients, 8 males and 7 females, aged from 1 to 11 years old, the medical indicators were within the normal range. There was no age difference between the groups (P>0.05) (Table 3.1).Diagnosis and treatment standards are in line "treatment of aplastic anemia in children recommendations. "[1]All patients in recovery period were not from blood transfusion.2 MethodsCD4+CD25int/highCD127low Treg cells in peripheral blood expression level were performed by Flow cytometry; extracted peripheral blood mononuclear cells, and extraction of total protein in the cells, using Western-blotting method to detect NFAT1, Foxp3 protein expression. The X-ray of experiment results were scaned by scanner.The average gray striped gray background of the image were analysised Quanity One software, the former minus the latter as the statistical analysis of value. Final statistics of the samples were the ratio of the gray values and the corresponding values of the reference.3 Statistics analysisAll data using SPSS 16.0 statistical software, all the original data are for normal distribution and homogeneity of variance test, the experimental data as mean± standard deviation (x±s) that the diversity of the mean compared with single factor analysis of variance (one-way ANOVA).Among groups using LSD-t method, the correlation between variables were analyzed using Pearson correlation analysis toα= 0.05 level for the test.Results1 Peripheral blood CD4+CD25+,CD4+CD25high,CD4+CD25+CD127low CD4+CD25highCD127low percentage of total CD4+ T cells of CAA group in the initial treatment period were lower than the control group (P<0.05); the expression of recovery period were higher than the initial treatment period (P< 0.05), while with the control group no significant difference (P>0.05).2 Peripheral blood mononuclear cells of Foxp3, NFAT1 protein expression levels of CAA group in the initial treatment period were 0.86±0.06,0.77±0.08; significantly lower than the control group (P<0.05); the expression level of recovery period were 1.20±0.26,1.11±0.12; significantly higher than the initial treatment period (P<0.05).3 Correlation Analysis(1) CD4+CD25highCD127low regulatory T cells and peripheral blood mononuclear cells Foxp3, NFAT1 protein expression showed a positive correlation in children of the chronic aplastic anemia (r=0.857,0.880, P<0.05).(2) Peripheral blood mononuclear cells Foxp3, NFAT1 protein expression was positively correlated in children of the chronic aplastic anemia (r=0.807, P< 0.05).Conclusion1 Peripheral blood CD4+CD25int/high CD127low Treg cells and NFAT1, Foxp3 protein expression of CAA group were decreased,possibly in the pathogenesis of chronic aplastic anemia play an important role, and the initial treatment period was lower than that of recovery period, it expected to assess the efficacy of aplastic anemia. 2 Foxp3 and NFAT1 protein levels were positively correlated, indicating that both in development and differentiation of Treg play a synergistic role. |
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