| Objective To analyse differential protein expressions of bone marrow CD34+ cells and m DCs(myeloid dendritic cells)in patients with SAA(Severe Aplastic Anemia)and healthy subjects by proteomics,in order to screen homologous proteins which activate immune response.Study on the expression level of homologous proteins,and the correlation with clinical data,and its influence on the m DCs,effector T cell and target cells.To investigate its role in the pathogenesis of SAA.To establish immune-mediated bone marrow failure model in mice,and to analyze the expression level of the protein in mouse model,further explore the initial immunic activation factors of SAA,and provide the experimental basis for the clinical application of immunosuppressants,reduce disease relapse.Methods The objects of the study are patients admitted to the department of hematology,Tianjin Medical University General Hospital from 4/2014 to 4/2017,including untreated SAA patients,remission SAA patients and normal controls.C56BL/6 mice,BALB/c mice and CBy B6F1 mice were bred in the animal laboratory of the Institute of radiation medicine,Chinese Academy of medical sciences.Part 1 To explore the substance of SAA patients immune activation.In our research group preliminary work,mononuclear cells from SAA patients and normal subjects were cultured in vitro differentiated into mature m DCs,sorting by flow cytometry,total protens were extracted,two dimensional electrophoresis separation of proteins,different proten components were identified.CD34+ cells were sorting by immunomagnetic beads,total proteins were abstracted,and labled by i TRAQ,the proteins were investigated by multidimensional liquid chromatography and tandem mass spectrometry,different proten components were identified.Homologous comparison of differential protein expressions simultaneously in m DCs and CD34+ cells between SAA patients and normal controls,to explore the probable antigenic substance leading to immune activation.Part 2 To examine the expression of screened differential proteins in SAA patients and normal controls.26 SAA untreated patients,22 SAA remission patients and 18 healthy controls were enrolled.Homogenic leukocyte immunoglobulin like receptor A isoform(LILRA)expression in m DCs,CD34+ cells,CD3+CD8+ cells were examined by flow cytometry(FCM).q RT-PCR and Western Bolt were used to detect the relative expression level of m RNA and the expression of LILRA in BMMNC,m DCs and CD34+ cells.Simultaneously,many parameters were examined,such as the quantities,proliferation and activation factor of m DC,CD3+CD4+/CD3+CD8+,Th1/Th2,the quantities,functions and cytotoxic factor expression of CTL,the data and SAA patient state were correlation analyzed.Part 3 To testify the activating effect of m DCs and the apoptosis effect of targeted cells by LILRA3 in vitro cell model.To knockout LILRA3 with RNAi(low expression group)in sorted m DCs and sorted CD34+/CD3-targeted cells of SAA patients and normal controls.m DCs and sorted CD8+MHC-I+ CTLs were co-cultured in vitro,the functions of m DC and CTL were examined.CD8+MHC-I+ CTLs and targeted cells were co-cultured in vitro,and the apoptosis of targeted cells were examined.Part 4 To establish a mouse model of immune-mediated bone marrow failure.The mice received non-lethal dose irradiation,bone marrow hematopoietic impairment can be further induced when the lymph node cells with minor histocompatibility incompatibility are infused.Complete peripheral blood counts and bone marrow cells morphology were examined.Pathological biopsy sample were obtained from liver,spleen and bone marrow.The expression of bone marrow LSK,the paired immunoglobulin-like receptors(PIRs)which homologous to human LILRs were examined by FCM.ResultsPart1: In the preliminary study,the differential expression of m DCs protein components in SAA patients and healthy subjects were observed,in the untreated SAA group,20 of which were up-regulated and 21 proteins were down-regulated.The differential expression of CD34+ cells protein components in SAA patients and healthy subjects were observed,in the untreated SAA group,54 proteins were up-regulated and 103 proteins were down-regulated.Homology and cross comparison of up-regulated differential proteins in the newly diagnosed group wrer made by BLAST.Compared with normal controls,the expression of LILRA3 in m DC cells of SAA patients increased;which highly homologous with LILRA5 which highly expressed on CD34+ cells,and combined with the same MHC-I ligand.q RT-PCR suggested that the relative expression level of LILRA3 m RNA in untreated SAA patients group(3.51±3.29)was significiantly higher than that of normal controls(1.37±0.96)(P<0.05)and remission SAA patients(2.45±1.77)was significiantly higher than normal controls(P<0.05).The relative expression levels of LILRA5 m RNA in SAA group were higher than that in normal control group,but the difference was not statistically significant.Part2: To examine the expression of LILRA in SAA patients and normal controls.The expression of LILRA3+ /CD34+ in untreated SAA patients group [(47.05±32.07)%] was significiantly higher than normal controls [(14.26±14.00)%](P<0.05)and remission SAA patients [(39.86±19.46)%] was higher than normal controls(P<0.05).The ratio of LILRA3+ /CD11C+HLA-DR+ in untreated SAA patients [(73.11±22.54)%] was significiantly higher than normal controls [(48.64±38.03)%](P<0.05),no significant difference between SAA remission patients group and normal groups.The relative expression of LILRA3 m RNA in m DCs of untreated SAA group(1.79±1.30)was higher than that in normal control group(0.77±0.72)(P<0.05).The level of LILRA3 protein in m DCs was significantly increased in untreated SAA patients group by Western Blot.The expressive level of LILRA3 m RNA was positively correlated with the proportion of LILRA3+ /CD11C+HLA-DR+(r=0.344,P<0.05).The expression of LILRA3+ /CD34+ was positively correlated with the proportion of LILRA3+ /CD11C+HLA-DR+(r=0.330,P<0.05)and negatively correlated with platelet count(r=-0.497,P<0.05)and hemoglobin(r=-0.427,P<0.05).Part3:The expresson of LILRA3 ligand HLA-A/B/C on CD8+ cells,showed no significant difference in three groups.After LILRA3 were knockdowned in sorted m DCs and CD34+ cells,the ratio of CD178(Fas L)+ /CD3+CD8+ in untreated SAA patients group [(14.22±6.74)%] was significantly higher than that in LILRA3 low expression of m DCs of SAA and NC CTL co culture group [(4.67±1.60)%] and m DCs of SAA and NC CTL co culture group [(4.53±1.73)%](P<0.05).The ratio of CD95(Fas)+ /CD34+ in LILRA3 knockdowned CD34+ cells and NC CTL co culture group [(46.59±26.00)%] was lower than in untreated SAA patients group [(78.24±27.60)%],but there was no significant difference between the three groups(P>0.05).The apoptosis rate in LILRA3 knockdowned CD34+ cells and NC CTL co culture group [(36.03±22.09)%] was lower than in untreated SAA patients group [(39.86±15.91)%],but there was no significant difference between the three groups(P>0.05).The activity of CTL in LILRA3 knockdowned m DC cells and NC CTL co culture group [(1.39±0.25)%] was significantly lower than in untreated SAA patients group [(2.27±1.76)%](P<0.05).Part4: A mouse model of immune-mediated bone marrow failure was established.The mice in the experimental group had received Cesium-137 5.0-6.0 Gy(gamma ray dose rate 1 Gy/min),4-6 hours after irradiation lymphocyte suspension was injected into CBy B6F1 mice(H2b/d).After fourteen days peripheral blood was tested show that the experimental group of mice pancytopenia rate was 100%,the mortality rate was 15%,in severe pancytopenia,RET and neutrophil absolute value decrease was significantly lower than that in normal control group and TBI irradiation group.Biopsy showed bone marrow hyperplasia was significantly decreased,the effective volume of hematopoietic 15-20%,granulocyte and erythrocyte lines decreased significantly,no megakaryocytes were found.In experimental group,bone marrow count,CFU-GM,CFU-E,BFU-E,CFU-GEMM colony forming unit count,HPCs and HSCs counts were significantly lower than the TBI irradiation group and normal control group.The mice in the experimental group met the criteria of the AA animal model.The expresson of PIR-A/B+ on CD34+ cells,showed no significant difference in three groups.The ratio of PIR-A/B+CD8+/CD3+ in experimental group [(2.89±1.57)%] was significantly lower than the TBI irradiation group [(19.63±12.50)%] and normal control group [(23.77±10.58)%](P<0.05).The ratio of PIR-A/B+/CD11c+ in experimental group [(50.08±20.65)%] was significantly lower than normal control group [(72.03±17.55)%](P<0.05).Conclusions In SAA patients,the increased expression of LILRA3 in m DC cells was highly homologous with increased expression of LILRA5 in CD34+ cells.Both LILRA3 and LILRA5 are type I transmembrane glycoprotein,and combine with the same MHC-I ligand that suggest LILRA may have critical effects in the immunic pathogenesis of SAA patients.Our research has confirmed LILRA3 was highly expressed in both m DCs and CD34+ cells of SAA patients simultaneously.The expressive level of LILRA3 m RNA is positively correlated with LILRA3+ /CD11C+HLA-DR+.The expression of LILRA3+ /CD34+ is positively correlated with LILRA3+ /CD11C+HLA-DR+ and negatively correlated with platelet count and hemoglobin.In SAA patients,the up-regulated LILRA3 may be involved in the activation of m DCs and hyperfunction of CTLs.AA mouse model was successfully established.In the SAA group,the expression of LILRA3 was upregulated and hyperfunction.With effective treatment,the expression level of LILRA3 decreased.LILRA3 may have critical effects in the immunic pathogenesis of SAA,LILRA3 may be regarded as potential new therapeutic targets of SAA. |
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