The Establishment Of A New Model Of Inflammatory Muscle Disease And The Research Of The Pathogenesis Of Polymyositis By OX40/OX40L

Part I The building and expression of myosin binding protein C prokaryotic expressionOBJECTIVE: To construct a human rapid fibronectin-binding protein C(MYBPC2)fusion protein expression vector,to efficiently express and purify p ET28a-My BPC2 fusion protein,and to provide a basis for further study of the establishment of inflammatory myopathy animal model.Methods: Human MYBPC2 c DNA was used as a template,and the target gene was obtained by PCR amplification with F and R primers.The target gene was cloned into the p ET28a(+)expression vector with Nco I / Xho I.The expression protein p ET28a-MYBPC2 was induced by IPTG.Chromatographic purification,dialysis renaturation,SDS-PAGE analysis and identification.Results:The expression conditions were optimized and the induction conditions were adjusted to 26 degrees.The target protein was expressed in the supernatant and the inclusion body.Therefore,the target protein was purified by Ni affinity chromatography.Conclusion: This method can efficiently prepare human MYBPC2 protein fragment,which provides a good foundation and method for the study of animal model of new experimental inflammatory myopathy.Part II The establishment of a new model of inflammatory muscle diseaseOBJECTIVE: To immunize mice with recombinant human myosin-binding protein C(MYBPC2)to obtain autoimmune myositis model.To explore the conditions and methods of immunizing mice with low dose protein.Methods: After fully emulsification of MYBPC2 and full foster-adjuvant,the C57 BL / 6 mice were immunized in the back of the back of the C57 BL / 6 mice,and the abdominal cavity was injected with pertussis toxin.Divided into high and low dose of fusion protein to immune mice respectively,before immune and after 7 d,14 d and 21 d and 28 d testing its strength,and at 7,14,21,28,muscle tissue by HE staining and immunohistochemical staining observation major histocompatibility complex(MHC)-? class molecules and inflammatory cells infiltration and other pathological changes.Statistical analysis was carried out by rank sum test.Results:(1)and high dose of fusion protein in the study of existing reports(600 ug/only),compared to low doses of fusion protein immune mice also can obtain more satisfactory animal model of inflammatory myopathy,including muscle damage in mice in 14 days the most significant,visible muscle tissue degeneration and necrosis of muscle fiber in model mice,muscle fiber atrophy,and inflammatory cells infiltration.The mouse model group muscle endurance 54.49 s,decreased significantly compared to the control group135.40(U=8.00,P<0.05)Model group MHC-? type molecular positive,muscle fiber surface between muscle fibers and blood vessels around the regional visible on CD4 + and CD8 + T cells.Conclusion: Use of low dose of fusion protein successful induction of inflammatory myopathy animal models and animal model of low mortality,animal models and human inflammatory myopathy have similar clinical and pathological characteristics of muscle,and for the further study of myositis pathogenesis and related treatment provides a new tool and economic security.Part III The study of the pathogenesis of inflammatory myopathy by OX40 / OX40LObjective: to study the expression of OX40 / OX40 L in muscle tissue and peripheral blood of patients with multiple myositis and the inflammatory response of mouse models.Methods: this study using immunohistochemical technique analysis of CD8,CD20,OX40 and OX40 L expression in the patients with muscle tissue,peripheral blood in patients with flow cytometry detection technology research the expression of OX40 / OX40 L situation,and using the method of ELISA test model the expression of serum cytokines in mice to observe inflammation and OX40 / OX40 L raised.Results:(1)the expression of ox40/ox40 l in the peripheral blood of patients with multiple myositis was raised;(2)OX40,OX40 L are found in inflammatory cells in muscle tissue of patients with polymytis.(3)the serum cytokines of the mice were raised higher than before.Conclusion: it is proved that ox40/ox40 l co-stimulated molecules are involved in multiple myositis and play an important role in the toxicity of myofibroblasts.