Study On The Effect Of Immune Intervention And Molecular Mechanism Of B7-1 Chimeric Antibody Treatment Lupus Nephritis Model Induced By Pristane

B7-1 molecule(CD80), a kind of costimulatory molecule, mainly expresses on antigen presenting cells(APC) surface, which is the receptor of CD28 molecules.Aft er the combination of CD80-CD28, it may mediate T cell activation, proliferation and differentiation, and thus stimulate the body’s immune response. B7-1 molecule expresses stablely under normal physiological conditions, to provide co-stimulatory signals for T cell activation, to maintain the survival of T cells. When B7-1under low expression, it will lead to T cell abnormalities, resulting in T cell anergy or apoptosis, causing the body sick. Conversely, B7-1 high expression will make excessive activation of T cells, causing autoimmune diseases.Autoimmune disease(AID) will lead to tissue damage by its own disease, mainly for T, B lymphocyte over-activation. Systemic lupus erythematosus(SLE) is a common autoimmune disease, in which SLE Lupus nephritis is the most common and most severe complications, one of the main causes of death in patients with SLE. Numerous studies showed that the expression of SLE patients in vivo B7-1 molecules significantly higher than healthy people, suggesting that B7-1 / CD28 costimulation signal plays an important role in the pathological process in SLE.Blockade the B7-1 / CD28 signaling pathway will reduce the body’s immune response. But traditional murine monoclonal antibody as rat source is high???, easy to cause human anti-mouse antibody response(HAMA), limiting its application. Chimeric antibodies using genetic engineering technology design is a hu manized recombinant antibodies, the application of recombinant DNA technology after the C region gene of murine monoclonal antibody V region genes with human Ig G stitching, inserting the appropriate vector, transfects antibody molecules corresponding host cell expression. Chimeric antibody contains the murine Fab fragment and the Fc portion of human origin, humanized portion reaches 70 percent, greatly reducing the HAMA response, extending the half-life period. Such antibodies on the one hand with the ability of murine monoclonal antibody Fab fragment binds with high affinity to an antigen, on the other hand its Fc portion of human origin in the patient but also mediate ADCC(antibody-dependent cell-mediated cytotoxicity), CDC(complement dependent cytotoxicity) and immune function.The aim is use of chemical methods to treat mouse lupus nephritis model and evaluate its biological basis for the the use of B7-1 chimeric antibody to mouse model intervention by immunology, serology, pathology and other indicator s, investigate B7-1 human-mouse chimeric antibody blockade B7-1 / CD28 signaling in mouse models of lupus nephritis pathological damage reversal effect against these diseases in order to find more efficient, safe, convenient administration antibody drug.1. Preparation and characterization of B7-1 human-mouse chimeric antibodyObjective: To prepare chimeric antibody B7-1 and identify their biological characteristics. Methods: The supernatant of B7-1chimeric antibody-producing cell lines were collected; antibodies were purificated by protein G affinity chromatography; recognition chimeric antibody to human and mouse B7-1 molecular by flow cytometry analysis. Results: protein the yield of chimeric antibody was 2.5mg / L; B7-1 chimeric antibody could recognize the human B7-1 gene transfected mouse fibroblasts strain L929-B7-1, lymphoma cell line Daudi, the mouse spleen cellswere 95.1%, 96.6% and 51.7% respectively. Conclusion: B7-1 chimeric antibody can specifically recognize B7-1 molecular expressed on cell surface.2. Lupus nephritis model establishment and biological identificationObjective: to establish the lupus nephritis model miceand to identify its immunological, serological, morphological and other biological changes. Methods: 6-week-old female C57 BL / 6J mice were randomly divided into two groups, the model group were administrated Pristane each 0.5ml by intraperitoneal injection, the control group received an same volume of saline. every-two-week serum ANA and anti-ds DNA antibody titers were detected,, protein urinary were detected monthly antigen presenting cells, plasma cells, activation of T cells of mice spleen cells were analyzied by flow cytometry kidney Pathological changes immunohistochemical analysis, transmission electron microscopy were also investigated. Results: Aftetwo months, serum anti-ds DNA antibodies rate of 60%, ANA positive rate was 90%; sfour months post induction, 80% of the mice urine protein reached++ ~ +++;flow cytometry analysis showed that the model mice spleen phagocytic cells, dendritic cells, granulocytes activation significantly increased(P <0.05), B cell surface CD21, CD80, CD86 molecule expression levels was significantly higher in model group(P <0.05), the T cell surface CD4, CD25, CD28, CD152 molecule expression was significantly higher(P <0.05); kidney renal biopsy shows pellets volume weight increased, inflammatory cell infiltration and tubular mild hyperemia, immunofluorescence staining showed a large number of immune complex deposition on glomeruli; TEM showed dense deposit obvious glomerular basement membrane thickening serious. Conclusion: Pristane induced mouse model of lupus-like nephritis were established.3. Immune intervention effect and molecular mechanisms study of B7-1 human mouse chimeric antibody against murine lupus nephritis modelObjective: To investigate the reversal effects of B7-1 human- mouse chimeric antibody blocking B7 / D28 signaling in mouse models of lupus nephritis pathological damage. Methods: according to the method described above lupus-like nephritis mouse model, mice were randomly divided into 3 groups:ⅠB7-1 antibody intervention group with human- mouse chimeric antibody administered intravenously through orbital vein, Ⅱthe positive control group was injected with immunosuppressant CTX, Ⅲmodel group injected with human isotype Ig G. then the monthly Urine protein, serum ANA and anti-ds DNA antibody titers,·· the mice were sacrificed, spleen cells were analyzied by flow cytometry to test the multiple immune-related cell maeker activation, and kidneys histopathologyic analysis, immune complex(IC) testing and TEM were performenced. Results: After the antibody intervention, urine protein concentration gradually reduced to the ++ ~ +++ to ± ~ ++, while the serum ANA and anti-ds DNA antibody fluorescence intensity was significantly reduced compared with model group with a statistically significant difference(P < 0.05).FCMresults showed that the intervention group antibody spleen phagocytic cells, dendritic cells, granulocytes activation was significantly lower than the model group(P <0.05), B cell surface CD80, CD86 molecule expression level model in the intervention group was significantly higher than antibody(P <0.05), T cell surface CD4, CD25, CD152 molecules expression model group than antibody intervention group were statistically significant(P <0.05); kidney HE staining showed that the intervention group antibody glomerular inflammatory cells invasion and tubular congestion and other symptoms were improved significantly. Immunofluorescence staining antibody antigen-antibody complexes in the intervention group of the fluorescence intensity was significantly reduced. transmission electron microscopy showed the electron dense deposits on reducing the glomerular basement membrane thickness becomes uniform in antibody intervention group compared with the model group,. Conclusion: B7-1 chimeric antibody could reduce the production of autoantibodies and exert reverse effect on the pathological damage caused by autoimmunity disease by inhibiting the B7-1 / CD28 signaling pathway to down-regulate the body’s immune response.