| Objective:To investigate immature dendritic cells loading autoantigen induced immune tolerance,to explore new methods of active immunization to prevent and treat SLE.Methods.1.ImDC extraction and culture:4～6 weeks SPF level Balb/c mice,extracted bone marrow cells,sterile culture.Observed the the changes of morphology and quantity under the inverted phase contrast microscope.Marked with monoclonal antibodies,identified cells by fluorescence microscopy,flow cytometry;examed the imDCs phagocytosis autoantigen composition by immunohistochemistry,transmission electron microscopy;studied imDCs loading autoantigen induced immune tolerance by flow cytometry,mixed lymphocyte reaction and ELISA.2.ImDC induced immune tolerance in vitro:imDCs loading autoantigen induced immune tolerance by flow cytometry,mixed lymphocyte reaction and ELISA.3.The establishment of mouse model of lupus:select 4～6 weeks SPF level Balb/c mice,all 30 mice were divided into 3 groups(sheared toes division),10 mice in each group were with the same genetic background.Extracted nucleoprotein as antigen from the same strains of Balb/c mice,immunized with a total of 4 times immunization(three weeks interval) by intramuscular injection.V1 group was injected with the nucleoprotein;V2 group was injected with the same volume PBS;V3 group was the control group.Analysised the success rate of modeling by detecting the mouse 24h urinary protein,serum ANA,anti-ds-DNA antibody,and direct immunofluorescence of the mouse kidney.4.ImDC loading autoantigen immuned the mouse model of lupus:select 4～6 weeks SPF level Balb/c mice,each group 10 mice with the same genetic background.S1 group was injected with nucleoprotein;S2 group injected imDCs loading antigen that derived from the same genetic background Balb/c mouse bone marrow through tail vein,one week before each immunization;S3 group was injected with the same volume PBS;S4 group was the control group.Observed the efficacy of immune tolerance,induced by imDCs loading autoantigen by detecting the mouse 24h urinary protein,serum ANA,antids -DNA antibody,and direct immunofluorescence of the mouse kidney.Results:1.33D1-positive cells accounted for 90.6 percent,meaning that the cultured DCs to achieve 90.6%purity.2.Proved imDC cytoplasmic debris appears in brown,nucleus were still blue by immunohistochemistry,transmission electron microscopy.3.DCs0 expressed MHCⅡfor(46.53±3.66)%,CD86 for(39.17±3.23)%; DCs1 expressed MHCⅡfor(31.53±4.07)%,CD86 for(20.07±1.60)%by flow cytometry detecting the surface markers of DCs.The expressions of MHCⅡand CD86 in DCs0 were lower than those of DCs0.The difference of MHCⅡwas with statistical significance(P＜0.01),the difference of CD86 was also with statistically significant(P＜0.05).Cultured with splenocytes of the same genetic background of Balb/c mice,the inhibition rate of DCs1 was higher than DCs0 by MTT,with statistically significant difference(P＜0.05).Detected the secretion of cytokine by ELISA,DCs1 secretion of IL-4 was higher than DCs0,and the difference was with statistically significant(P＜0.01);secretion of IFN-γwas less than DCs0,the difference with no statistically significant(P＞0.05).4.The 24h urine protein of V1 group was(702.10±3.81)mg/ml, significantly higher than V2 group(40.50±0.97)mg/ml and the V3 group (38.50±3.21) mg/ml.V1 group was significantly different with the other two groups,while the V2 and V3 groups were with no significant differences (F=170391.37,P=0.000);The anti-ds-DNA antibody of V1 group was (50.52±0.74)pg/ml,significantly higher than V2 group(8.60±0.63)pg/ml and the V3 group(8.43±1.30)pg/ml.V1 group was significantly different with the other two groups,while the V2 and V3 groups were with no significant differences(F=6719.201,P=0.000);The ANA of V1 group was(22.92±1.73)ng/ml, significantly higher than V2 group(7.88±1.08) ng/ml and the V3 group (8.19±0.84)ng/ml.V1 group was significantly different with the other two groups,while the V2 and V3 groups were with no significant differences (F=457.452,P=0.000);there were IgG,IgM immune complex deposition in V1 group glomerular;in V2,V3 group no glomerular profile only weak non-specific fluorescence could be seen.5.The 24h urine protein of S1 group was(875.10±9.04)mg/ml,higher than the S2 group(368.50±7.00)mg/ml,significantly higher than S3 group (51.40±5.58)mg/ml and S4 group(49.30±4.60)mg/ml.S3 and S4 groups were with no differences;there were statistically significant differences between S1, S2,S3 and S4 groups(F=33164.524,P=0.000);the anti-ds-DNA antibody of S1 was(55.52±3.04)pg/ml,higher than the S2 group(27.90±4.35)pg/ml, significantly higher than S3 group(9.58±1.94)pg/ml and S4 group(8.92±3.31) pg/ml.S3 and S4 groups were with no differences;there were statistically significant differences between S1,S2,S3 and S4 groups(F=445.93,P=0.000);the ANA of S1 group was(23.37±1.57)ng/ml,higher than the S2 group(17.24±1.39) ng/ml,significantly higher than S3 group(8.18±1.27) ng/ml and S4 group (8.16±1.39)ng/ml.S3 and S4 groups were with no differences;there were statistically significant differences between S1,S2,S3 and S4 groups(F=278.619, P=0.000);the proportion of CD4~+ splenocytes of S2 group increased,the proportion of the spleen CD8~+ lymphocytes reduced,the ratio of CD4~+CD25~+ cell increased.S1 group had glomerular IgG,IgM immune complex deposition,glomerular profile could be seen.There was weak fluorescence in S2 group;no glomerular profile,only weak non-specific fluorescence could be seen in S3,S4 groups.Conclusion:1.ImDC could swallow autoantigen,which inhibited imDCs further development,maintained the immature state of imDCs,while induced immune tolerance to autoantigen.2.By extracting nucleoprotein as antigen from the same genetic background of Balb/c mice,established mouse model of lupus successfully in inbred Balb/c mice,its pathological state was similar to human SLE.3.ImDC loading autoantigen could induce the mouse model of lupus with the same genetic background to immune tolerance,which prevented or mitigated the role of disease.
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