Study On The Hematological Characteristics In Mice With Integrin Beta3 Gene Deficiency

Integrins are a wide range of cell-adhesion receptors,mainly composed of the α subunit and the β subunit.Among them,the integrin β3 subunit can form integrin αIIbβ3 with the αIIb subunit,but also can form integrin α?β3 with the α? subunit.Integrin αIIbβ3 is the most abundant receptor on the surface of platelets and megakaryocytes,which mediates platelet bidirectional signaling.Currently,it is known that about half of the human platelet antigen(HPA)is localized in the integrin β3 cytoplasmic segment.The complete or partial deletion of a IIbβ3 receptor can cause glanzmann thrombasthenia(GT).The patients with GT show defects in platelet aggregation and clot retraction,prolonged bleeding time.β3-/-mouse has been widely used as a GT model,but its’ hematological properties have not yet been fully elucidated.Fetal and neonatal alloimmune thrombocytopenia(FNAIT)is a serious hemorrhagic disease,mainly due to fetal and maternal platelet phenotype dismatch.The main risk of FNAIT is intracranial hemorrhage,which causes high mortality rate.At present,the pathogenesis and treatment of FNAIT is still incomplete.Objective: 1.To observes the hematological characteristics of integrin β3-/-mice.2.To establish FNAIT model using β3-/-mice to lay a foundation for the research of FNAIT.Methods: 1.To generate and identify integrin β3 knockout homozygous mice(β3-/-);2.To test the expression of integrin β3 in platelet by flow cytometry and Western blot;3.Blood of β3-/-mouse was collected by the heart puncture,and then blood count,blood smear and Swiss staining were made;4.The spleen weight was counted.Spleen and bone marrow tissue sections were made,Chronic hemorrhagic model was simulated by regular blood letting in β3 +/+ mice;5.The characteristics of platelets in β3-/-mice were explored by fibrinogen binding assay,spreading assay,adhesion on different medium,and thrombus formation under shear rates;6.β3-/-pups were observed to determine whether there is subcutaneous hemorrhage,and whether tail bleeding time is prolonged;7.β3-/-female mice were immunized with β3+/+ mouse platelets in twice a week with a transfusions of 109 platelets every time.Sera were prepared by centrifuging,and detected whether there is anti-integrin β3 antibody by flow cytometary after immunization;8.The immunized β3-/-female mice were mated with β3+/+ male mice to establish a model of fetal and neonatal alloimmune thrombocytopenia(FNAIT).Results: 1.Mouse genotype was identified successfully by PCR;2.The results of flow cytometry and Western blot showed that the platelet surface of β3-/-mice didn’t express integrin β3 subunit;3.Embryo β3-/-mice were more likely to bleed than β3+/+ mice.The bleeding time of β3-/-mice after tail-cutting was prolonged.The blood cell count indicated that the β3-/-mice were anemia.The peripheral blood smear showed microcytic hypochromic anemia;4.The spleen of β3-/-mice was significantly larger than that of β3+/+ mice.Spleen enlargement in chronic hemorrhagic model mice.The result of immunohistochemistry from spleen revealed normal expression of CD3 and CD19,but elevated expression of CD71 suggesting that the spleen enlargement of β3-/-mice is associated with extramedullary hematopoiesis;5.The function of fibrinogen binding,spreading and adhesion of β3-/-mouse platelets is obviously impaired;6.Anti-integrin β3 antibody in immunized β3-/-mice was successfully detected by flow cytometry;3.Immunized β3-/-female mice which were mated with β3+/+ male mice,were not easy to produce offspring,and were easy to miscarriage,and pregnant mice are prone to death.Conclusion: β3-/-mice are a good model of glanzmann thrombasthenia by investigating the hematological characteristics of β3-/-mice;The anti-integrin β3 antibody was detected in immunized β3-/-mice.The immunized β3-/-mice are not easy to get pregnant,prone to miscarriage,and die during pregnancy.The FNAIT mouse model was preliminary established.