The Proliferation Of T Cell Responses To Desmoglein 3 In Patients With Pemphigus Vulgaris

ObjectivePemphigus vulgaris(PV) is a potentially fatal blistering disease of the skin and mucous membranes characterized by the loss of intercellular adhesion(acantholysis) of keratinocytes due to binding of autoantibodies to the cell surface.The pathology shows cell lysis are acantholysis,the loss of intercellular adhesion,this changes occur are the direct cause of the existence of PV in patients with localized or circulating autoantibodies of IgG type,the antibody and keratinocytes(keratinocyte,KC) surface after the role of antigen molecules to the desmosome glycoprotein of a-glycoprotein desmoglein 3(desmoglein 3,Dsg3),ability to cause the loss of intercellular adhesion and cause the occurrence of PV.This can be through passive transfer of disease in patients with IgG to newborn mice and reproduce on humoral immunity in the occurrence of PV plays an important role in the process.With the technology in immunology and molecular biology technology,a growing body of research results show that the T lymphocytes in the pathogenesis of PV also plays an important role.In addition to secretion of various cytokines directly involved in the development of diseasesIn addition,Th2 cells in MHCâ…¡antigen recognition,B cell activation, antibody production,as well as target organ damage and other aspects of both play an important role.This experiment by flow cytometry detection of four fluorescent staining PV patients with Dsg3 stimulated peripheral blood T lymphocytes Th1 and Th2-type T lymphocyte changes in expression levels;The use of 5-BrdU incorporation assay CD4⁺ T lymphocyte proliferation,the preliminary study in patients with pemphigus vulgaris and its response to the pathogenesis of T lymphocyte activation and proliferation of the relationship between the two.Materials and Methods 1,Case and capital MaterialsPV patientsPV in patients with 12 people,all of China Medical University First Affiliated Hospital of dermatology patients.All cases were confirmed by clinical, histopathological and immunohistochemical pathology for pemphigus vulgaris; health 22 people as a normal control.Specimen collection and treatmentA study of peripheral blood 10ml,EDTA-k anticoagulant eight hours after the extraction of peripheral blood mononuclear cells(PMBC).Dsg3 SourceStable transfection of HELA cell line.The main reagentsLymphocyte separation medium,RPMI1640 medium,fetal bovine serum,Hyclone companies for products;phytohemagglutinin(PHA) for Sigma products;fluorescent-labeled antibodies(anti-human CD3-APC,anti-human CD4-PE^Cy5,anti-human IL-4-PE, anti-human IFN-γ-FITC),Cytofix/Cytoperm^TM Plus Fixation/Permeabilization Kit, FITC BrdU Flow Kit,BD GolgiStop^TM,24-hole culture plates were purchased from BD Company;Ni-NTA purification system,G418(Geneticin) for Invitrogen products; disposable filter(0.22μm) for Millipore products.2,The main instruments and equipmentFlow cytometry,FACS Calibur,ND1000(BD Company,USA);AKTA purifier (GM,United States);CO₂ incubator(Harris HW0301 T-VBA-type,Germany);low-temperature centrifuge(Hitachi,Japan);Clean Benches(China).Methods1,The access to Dsg3 autoantigen;2,Peripheral blood mononuclear cells(PBMC) of the extract;3,T lymphocyte activation and culture:4,Cultured T lymphocyte Th1/Th2 type determination; 5,5-BrdU incorporation assay self-reactive T lymphocyte proliferation.ResultsCultivate in each group after the Th1/Th2 type T lymphocyte results1,Dsg3 stimulated Th2-type PV patients was higher than the proportion of T lymphocyte without any stimulation of the negative control group;2,The experimental group and the negative control group Th1-type T lymphocytes no significant difference;With Dsg3:3,PV patients with the proportion of Th2-type T lymphocytes were significantly higher than normal people;4,PV patients with Th1-type T lymphocytes was lower than the proportion of normal people;Without any stimulation:5,PV patients with Th2-type T Iymphocytes were significantly higher than normal people;6,PV patients with Th1-type T lymphocytes were lower than normal people.5-BrdU incorporation assay of its response to the results of CD4⁺T lymphocyte1,PV patients with Dsg3 after stimulating the proliferation of CD4⁺T lymphocyte was significantly higher than normal;2,PV patients with Dsg3 after stimulating the proliferation of CD4⁺T lymphocyte without any stimulation was higher than the negative control group.Conclusion1,Dsg3 autoantigen in patients with PV can induce specific proliferation of T lymphocytes;2,Dsg3 at the stimulation,PV activity of T lymphocytes in patients with obvious advantages for the Th2 type,which inferred Th2-type T lymphocyte is probably the early onset PV important pathogenic factor; 3,This test has not yet been found in Th1-type T lymphocyte and contact between the incidence of PV.