Study Of The Immune Intervention Effect And The Molecular Mechanism Of Anti-human B7-1 Diabody On Lupus Nephritis Induced By Pristane

B7-1 molecules are significant surface costimulatory molecules expressed on antigen presenting cells(APC), its receptor CD28 is expressed on the T cell surface.CD28 binding molecules B7-1 transduced T cell activation positive costimulation. The signal promotes antigen-specific T cell proliferation, cytokine secretion, induction of effector T cells and prevents T cell apoptosis and other functions.Systemic lupus erythematosus(SLE) is a self-reactive T, B lymphocytes abnormal activation of autoimmune disease which produces antibodies and immune complexes and other characteristics(autoimmune disease, AID). The lesion involving the whole body, can cause chronic lesions and multiple organ and tissue function loss. Which especially lupus nephritis( LN) is the most serious complication and is the leading cause of death in patients with SLE. The study found, B7-1/CD28 signaling pathway plays an important role in the development of SLE disease, thus blocking the B7-1/CD28 signaling pathway for the autoimmune disease SLE intervention provides a theoretical basis.1. Using self-constructed engineered cell SA Ш to secrete anti-human B7-1bivalent and purify bivalent antibody.2.According to the pathological mechanism of injury in autoimmune disease SLE, Using pristane to induce C57BL/6 mouse model of lupus nephritis which is similar to human SLE and identified. 3. Application of anti-human B7-1 bivalent antibody to the disease model to study the intervention effect and its molecular mechanism. New targets and looking for new means of treatment of these diseases.I. Preparation and biological characterization of the mouse anti-human B7-1bivalent antibodiesObjective: To prepare mouse anti-human B7-1 bivalent antibodies and identifytheir biological characteristics. Methods: In vitro cell culture techniques of mass CHO cell culture supernatant was collected by centrifugation and ultrafiltration pretreatment on Profinity TM IMAC purified by nickel column; flow cytometry B7-1 bivalent antibody on the cell membrane type B7-1recognition; denaturing and non-denaturing conditions of SDS gel electrophoresis of the bivalent antibody bivalent sexual identification. Results: The supernatant of antibody protein yield 10mg/L; L929 cells transfected with the human B7-1 antibody gene, human Raji B cell lymphoma and the human T lymphoma cell binding rate was 99.3%, 97.9% and 9.5% respectively; the antibody in denaturing and non-denaturing conditions of SDS gel electrophoresis were27 kDa and 53 kDa protein bands purposes. Conclusion: Preparation of mouse anti-human B7-1 bivalent antibody.ⅡPristane induced mice lupus nephritis model establishment and identificationObjective: To construct pristane induced mouse model of lupus nephritis and biological identification of the model. Methods: Eight-week-old female C57BL/6 mice were randomly divided into two groups, the model group and negative control group were 10, making the module to the intraperitoneal injection of pristane 0.5mL/only, to a negative control group intraperitoneal injection of saline 0.5mL/only, casting time of 4months. 4 months, Albustix mouse urine protein dipstick urine protein; indirect immunofluorescence serum anti-double stranded DNA(ds-DNA) antibodies and antinuclear antibodies(ANA) content; Flow cytometric analysis of mouse spleen the APC, the percentage of T cells and B cells; direct immunofluorescence glomerular immune complexes(IC)deposition;glomerular pathology HE staining;glomerular TEM ultrastructural changes. Results: modeling four months, the mice were proteinuria,protein severity + ~ + +, urine protein content was 300mg/L ~ 1000mg/L; serum can be detected in the fluorescence intensity than the strong anti-dsDNA antibody and ANA;mouse spleen APC, levels of activated T cells and B cells was significantly higher(p<0.05); mouse glomerular mesangial deposition film IC serious; HE staining showed mouse glomerular hypertrophy, glomerular cysts irregular changes and severe inflammatory cell infiltration; TEM glomerular basement membrane thickening,glomerular basement membrane at a sharp angle, the visceral cells and the basement membrane visible among a large number of immune complex deposition. Conclusion:The establishment of pristane induced mouse model of lupus nephritis.Ш The study of molecular mechanisms of immune intervention effect B7-1bivalent antibodies in mice and lupus nephritisObjective: Using mouse anti-human B7-1 bivalent antibodies to block or weaken the B7/CD28 signaling pathway, pristane induced C57BL/6 mice model of lupus nephritis,to study the intervention effects and possible molecular mechanism research.Methods: The method according to the second part of the modeling, taking female C57BL/6 mice were randomly divided into four groups of five. Control group: reference antibody group at the same time giving the same amount of saline; model group:reference antibody group at the same time give an equivalent dose of mouse IgG isotype control; antibody group: Let the first injection time of the first day, in the first, 3,5,8,15,43,71,99 days were injected intravenously mouse anti-human B7-1 bivalent antibody10mg/kgw/only;CTX group: Let the first injection time of the first day, in the first 8, 22,29,43, 50,64,71,85,92 days, respectively abdominal injection CTX 60mg/kgw/only.Albustix mouse urine protein dipstick urine protein; indirect immunofluorescence serum anti-ds-DNA antibodies and ANA content; the percentage of flow cytometric analysis of mouse spleen APC, T cells and B cells; staining kidney pellets pathology; direct immunofluorescence glomerular immune complex(IC) deposition;glomerular TEM ultrastructural changes.Results: Antibodies mice urine protein content than the model group decreased significantly; serum antibody group of anti-dsDNA antibodies and ANA fluorescence intensity than the model group decreased significantly; antibody mice spleen APC, T cells and B cell activation levels were significantly lower than model group(p<0.05); HE staining antibodies mice showed glomerular hypertrophy,glomerular cysts irregular changes and inflammatory cell infiltration significantly reduced; antibody group small rat mesangial deposition film IC significantly reduced;antibody mice TEM glomerular basement membrane thickening significantly reduced visceral between cells and the basement membrane significantly reduced IC deposition.Conclusion: B7-1 bivalent antibody by B7/CD28 signal blocking or weakening reduces T, B cell abnormal activation, reducing the production of autoantibodies, immune complexes thereby reducing renal damage. Thus B7-1 bivalent antibody immune intervention can reverse the pristane induced mouse lupus nephritis pathological lesions,suggesting that the antibody has potential applications in the treatment of SLE disease.