Differentiation Of Cord Blood CD34+Cells In MG Patients With Thymus Transplantation In Nude Mice

Myasthenia gravis(MG) is a kind of organ specificity autoimmune diseases in which acetylcholine AchR antibody mediated and cellular immunity and complement participate. There are thymic hyperplasia and thymoma in70%～80%MG patients. The pathological characteristics of thymus of MG patients are existing T cell region and germinal center which rich in B cells of analogous lymph node structure. The autoantibody against AchR are found in MG patients thymus. Therefore, thymus has been regarded as the important place for B cells to produce autoantibodies.Thymus is an important central immune organs, is the place of mature T cells. MG patients with thymic abnormalities of abnormal differentiation of T cells is not fully understood. To further explore the abnormal thymus in T cell development and differentiation of the role of the thymus in this study, birth defects in nude mice, artificially transplant patients with normal thymus and MG thymus, nude mice to establish animal models of human thymus, Observed by transplanting umbilical cord blood CD34+stem cells in animal models of differentiation results to determine the MG patients with thymus in immune cell development and differentiation in the role. Methods1. MG patients MG patients diagnosed by clinical trials. Thymectomy pathological diagnosis of thymic hyperplasia type2. Thymus transplantation in nude mice model The MG hyperplastic thymus and congenital heart group thymus were cut about1mm×1mm×1mm size. In turn cut back skin of nude mice, muscle, fascia and other organizations, the left kidney exposed to nude mice, carefully cut the renal capsule, the thymus tissue implanted in the left kidney capsule sutured.3. Cord blood CD34+cells sort and transplantation Take the healthy full-term natural delivery after a maternity break the umbilical cord blood. Carefully add the umbilical vein to the surface of lymphocyte separation medium,2500r/min centrifugation for20minutes, drawing on mononuclear cell layer, flow optometry (FCM) detection of CD34+cells and other blood ratio.2weeks after thymus transplantation in nude mice, the freshly isolated cord blood CD34+cell suspension intravenously in mice, each mouse type2×105cells. Continue feeding for45days.4. Sources of human blood lymphocytes in nude mice60days after thymus transplantation, mice retinal blood0.5ml, lysis of red blood cells, PBS washed three times, were added to FITC2anti-human CD3and anti-human CD56monoclonal antibody PE2, FITC2anti-CD4and anti-human CD25for PE2double fluorescent labeling, FITC labeled CD19, CD161, HLA2ABC, HLA2DR single fluorescence labeled monoclonal antibody.To the same type of fluorescent antibodies for the control of the same,4℃, incubated for30minutes, PBS washed2times, resuspended to200μl on flow optometry.Results1. Human thymus in nude mice animal modelThe nude mice of MG patients thymus transplant survived. After2weeks in thymus transplantation, cord blood CD34+cells were injected in mice tail vein. Histological observation showed that transplantation of thymus tissue in the renal capsule, renal parenchyma with clear boundaries, a transplanted thymus cortex and medulla of normal structure, filled with a large number of cortical and medullae thymocytes, Detected by immunohistochemistry, CD3+positive cells widely distributed in the transplantation of thymus tissue.2. The rate of lymphocytes in MG patients thymus-nude miceThe rate of CD3+, CD4+, CD19+, CD161+cells was significantly higher in MG thymus and CD34+cells combine transplantation than the control group. Congenital heart group CD3+, CD19+cells ratio and MG proportion of CD4+cells increased more significantly. MG thymus transplantation group CD3+CD56+cells and CD4+CD25+cells was significantly higher than the single cord blood transplantation.ConclusionsThe MG patient thymus survive in BALB/c nu/nu nude mice and the transplantation restore thymic function. It can promote in vivo proliferation of cord blood stem cells transfused in unde mice. The human thymus-nude mice model can been used in studying differentiation of stem and lymphocytes.