| Systemic lupus erythematosus (SLE) is an abnormal disease that characterized byautoantibodies, which are produced by autoreactive immune cells. It can affect the body,causing chronic destruction and loss of function of each organ joint, skin, kidney, heart,lung, blood vessel and tissue irreversibility. Lupus nephritis (LN) is the most commonand serious complications, it is also the major causes of death. The cause of the diseaseis not clear yet, and may be associated with genetic and environmental factors.B7-2molecule belongs to one of the immunoglobulin superfamily members, and isan important costimulatory molecule on the surface of antigen presenting cells (APC). Itis Combined with CD28expressed on the surface of T cell, mediated T cell activation,proliferation, differentiation and immune effect. The lack of this signal, T cells willremain no response (Anergy) or immune tolerance (Tolerance) and even causeprogrammed cell death (Apoptosis). The study found that B7-CD28costimulatorysignals involved in the occurrence and development of SLE, and play an important rolein it. Anti-B7monoclonal antibody can block or weaken the B7-CD28signal, therebysuppressing the immune response, so the application of anti-B7-2antibody blocking orweakening the B7-CD28pathway is expected to provide a new method for biologicaltreatment of SLE.This research will establish the chronic graft-versus-host disease (cGVHD) murinelupus nephritis model that similar to human SLE. Anti-human B7-2monoclonalantibody will be used to intervene this model, then we will examine the reversal effectand explore its possible molecular mechanism by immunology, serology, kidney tissuemorphology and other aspects of the indicators. 1.Preparation and characterization of anti-human B7-2monoclonal antibodyObjective: to prepare anti-human B7-2monoclonal antibody and identify itsbiological characteristics. Methods: utilizing the hybridoma cell strain1D1which issuccessfully secreting stable anti-human B7-2monoclonal antibody; preparingmonoclonal antibody by inducing ascites in vivo; purifying the antibody by ProteinGimmune affinity chromatography; analyzing whether the antibody can recognize B7-2molecule of different cell types by flow cytometry. Results: after induced mouse ascitesin vivo of anti-human B7-2monoclonal antibody, the positive rate of ascites formationwas about80%;3ml ascites was obtained from each mouse; anti-human B7-2monoclonal antibody was3mg/ml after the ascites were purified by Protein G immuneaffinity chromatography; the anti-human B7-2monoclonal antibody can recognizemembrane B7-2molecule on human B7-2gene transfected mouse fibroblast cell lineL929-B7-2, Daudi and mice spleen cells with the positive of99.3%,96.7%and61.2%.Conclusions: anti-human B7-2monoclonal antibody can specifically recognizemembrane B7-2molecule of different cell types.2.Development and pathological evaluation of murine lupus nephritis model ofcGVHDObjective: To evaluate the murine lupus nephritis model by immunology,Serology and nephrology methods. To identify the reliability of this model as SLEanimal ones. Methods:(C57BL/6×BALB/c) F1hybrids are used as recipients ofBALB/c donor. Single-cell suspensions from donor’s spleen were injected4times at3days intervals. One week after that, immunofluorescence staining and flow cytometrywere used to analyze percentage of APCs and antibody produced cells among murinespleen cells. To detect anti-dsDNA antibody and ANA at2weeks intervals from serum;to test the proteinuria dynamic variation every4weeks;12weeks after the injection, thekidneys were slided and stained with HE to observe glomerulonephritis, depositions ofimmunoglobulin were examined by direct immunofluorescence staining on kidneysections. Electron microscopic was used to observe ultrastructure of glomerulus’schange. Results: one week after the induction of the model, the populations of CD11b+,CD11c+, GR1+cells were increased significantly（P<0.05）, meanwhile, the expressionsof CD21, CD23, CD80, CD86on B220+cells were also increased（P<0.05）.Anti-dsDNA antibody was positive after2weeks and ANA was observed after4weeks.After12weeks,80%experimental murine has proteinuria(++~++++), both optical microscopy and immunoflorescence of kidney sections indicated typical evidence ofglomerulonephritis. Electron micrographs showed electron dense doposits werelocalized in subepithelial lesions of glomerular basement membrane. Conclusions: Thismurine lupus nephritis model of cGVHD has similar symptom to human SLE and is areceivable model for related research.3.Study of the immune intervention effect and the molecular mechanism ofanti-human B7-2antibody on lupus nephritisObjective: To investigate the immune intervention effect and the possiblemolecular mechanism of anti-human B7-2monoclonal antibody on murine lupusnephritis model by blocking B7/CD28signaling pathway. Methods: according to themodel method of the second part, the female BCF1mice were randomly divided into3groups, antibody intervention group, model group and control group,15mice in eachgroup. The intervention group were intravenously injected anti-human B7-2monoclonalantibody (clone1D1)8mg/kg at the1,3,5,8and15d respectively after the lastinjection of lymphocyte, then1times injection every month. Model group at the sametime were given the same dose of mouse Ig isotype. Analyzing the percentage of theMacrophages (CD11b+), the dendritic cells (CD11c+), the neutrophil (Gr1+) in thespleen and the expression of CD21, CD23, CD80and CD86molecules on antibodyforming cell(B220+) surface, anti-dsDNA antibody, ANA content, the content ofProteinuria, immune complexes, glomerular morphology and ultrastructure change ofthe three group. Results: the expression of CD11b+, CD11c+and GR1+in spleen cellsand CD21+, CD23+, CD80+and CD86+in B cells in the intervention group were lowerthan the experiment group(p<0.05) by immunofluorescence and flow cytometryanalysis, suggesting that anti-B7-2antibody can inhibit immune response of the body byblocking the B7/CD28signaling pathway;90%mice in the model group can detect theexpression of anti-dsDNA antibody and ANA in the serum, the intervention group only40%, and antibody titers of the intervention group was significantly lower than that inthe model group;12weeks later,80%mice of the model group had proteinuria, and theprotein content is++~++++,20%mice of the intervention group were detected thepresence of proteinuria, protein content is+~++; kidney tissue HE staining showed thatthe renal glomerulus swelling, infiltration of lymphocytes and other inflammatorytypical changes of the model group, the intervention group glomerular volume increased slightly, a little lymphocytic infiltration, the volume of capillary lumen and ballooncavity were basically normal. Compared the two groups, renal pathological changes ofthe intervention group was lighter than the model group; the model group was foundglomerular IC deposition by fluorescence microscopy, compared with the model group,the IC deposition of intervention group was less than that of the model group;ultrastructure examination showed the basement membrane of the model group wassegmental thickened, a lot of subepithelial electron dense deposits were existed, thebasement membrane of the intervention group is normal, pathological change is lightercompared with the model group. Conclusions: anti-human B7-2monoclonal antibodycan inhibit the abnormal activation of immune cells by inhibition of B7/CD28signal,and the production of autoantibodies were reduced to alleviate renal function injury.Therefore, immune intervention of anti-B7-2monoclonal antibody can reversepathological injury of lupus nephritis, suggesting that it has a potential therapeutic effecton this disease. |
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