Study On The Mechanism Of Immune-related Damage On The Bone Marrow Hematopoiesis In Severe Aplastic Anemia

ObjectiveApplication of isobaric tags for relative and absolute quantification (iTRAQ) technology, the proteins of CD34+cells in bone marrow for patients with severe aplastic anemia (SAA) and normal controls were analyzed. To obtain differential proteomics profiles of CD34+cells in bone marrow for SAA specificity. To preliminary screen the autoantigen which may cause immune system hyperfunction in SAA, providing a theoretical basis for further elucidating the immune pathogenesis of SAA.To detect the serum levels of interleukin(IL)-17in peripheral blood of patients with SAA, to analyze correlation between IL-17and the T lymphocyte subsets, dendritic cell (DC) subsets, regulatory T cells (Treg) and the blood routine, to explore the relationship between IL-17and immunological mechanism for SAA, providing a theoretical basis for finding new therapeutic approaches to SAA.MethodsUntreated SAA patients, remission SAA patients, as well as normal controls in Tianjin Medical University General Hospital from2013.1to2014.2were enrolled in this study. Part I To establish differential proteomics profiles profiles of CD34+cells in bone marrow for SAA. CD34+cells in bone marrow for29cases of recovery SAA patients and10normal human were purified by immunomagnetic cell sorting, total protein were extracted and were processed enzymolysis, labeled sample by iTRAQ reagent, then analyzed the protein of sample by multidimensional liquid chromatography tandem mass spectrometry of Q Exactive, identified proteins by application of database, compared the different expression of proteins between SAA patients and normal controls, to preliminary screen the autoantigen which may cause immune system hyperfunction in SAA. Part II To investigate the role of interleukin IL-17in the pathogenesis of SAA patients. IL-17level in peripheral blood serum for25cases of SAA untreated patients,15SAA recovery patients and10normal controls were detected with cytometric beads array(CBA). The technology of monoclonal antibody labeling and flow cytometry were used to detect T lymphocyte subsets (CD4+/CD8+ratio), DC subsets (myeloid dendritic cells/lymphoid dendritic cell ratio, the ratio of mDC/pDC), regulatory T cells (Treg) ratio in different groups. Analyzed the correlation between IL-17levels in serum and T subset, DC subsets, Treg ratio and the blood routine.ResultsPart Ⅰ Expression of differential proteomic profiles for CD34+cells in SAA bone marrow were obtained. The protein confidence>95%and identification of peptide protein false positive rate<1%were limited,15120peptides and3208protein were identified. The Ratio of different proteins (SAA group/normal group) greater than2.794and less than0.323and the confidence limit greater than95%were limited,157different proteins with high confidence were identified, of which54up-regulated proteins,103down regulated proteins. The physiological function of proteins involved cell apoptosis, cell cycle, RNA splicing and metabolism, protein modification and transfer, the ubiquitin proteasome mediated protein degradation.Part Ⅱ The level of serum IL-17in untreated patients was higher than that in recovery patients and normal controls[(17.07±15.18)pg/ml vs (7.09±3.84)pg/ml and (3.53±2.08)pg/ml,p<0.01], and there also was significant differences between latter two groups(p<0.05). The ratio of CD4+/CD8+was (0.32±0.08)in untreated patients, which was lower than that in recovery patients [(1.11±0.31), P<0.01] and normal controls[(1.07±0.26),p<0.01]. The ratio of mDC/pDC was (3.16±0.55) in untreated patients, which was higher than that in recovery patients[(1.6±0.43), P<0.01] and normal controls[(1.43±0.38), P<0.01]. The percentage of Tregs in peripheral blood lymphocyte (CD4+CD25+CD127dim/PBL) was (0.8±0.31)%in untreated patients, which was lower than that in recovery patients[(1.78±0.69)%, P<0.01] and normal controls[(2.23±0.66)%, P<0O.01]. The level of serum IL-17in untreated SAA patients was positive related with mDC/pDC ratio (r=0.414, P<0.05), and that was negative related with CD4+/CD8+ratio (r=-0.421, P<0.05) and CD4+CD25+CD127dim Tregs/PBL(r=-0.65, P<0.01). There were significant negative correlations between serum IL-17and white blood cells in untreated patients (r=-0.689, P<0.01) and recovery patients (r=-0.64, P<0.05). Conclusions1. The proteome profiles of CD34+cells in SAA bone marrow were obtained successfully, and it was proved that iTRAQ marker tandem mass spectrometry for proteome of bone marrow cells for patients with SAA was an effective method. It was found that157abnormal expression proteins of CD34+cells in SAA bone marrow, the function of proteins mainly involved apoptosis, cell cycle, protein modification and transport, RNA processing and splicing, the ubiquitin proteasome system. We preliminary predicted autoantigen which may cause SAA immune "waterfall" activation.2. The level of serum IL-17in patients with SAA was increased significantly. After ATG plus cyclosporine A intensive immune suppressive treatment, the level of IL-17decreased but still higher than normal. And the level of IL-17in serum was related to the status of disease severity and immune function in patients with SAA. IL-17may be a new target in the treatment of SAA, and may be an index for assessment of curative effect and prognosis of the disease.