| Research background and purposeThe thymus is the central immune organs of the body, is the place for mature T cell differentiation. Formation in nude mice is due to mutations in congenital athymic hairless mice, with severe T cell deficiency and cellular immune dysfunction, the research in human tumors in nude mice, immune deficiency, anti-infection immunity and other aspects of a more extensive application. Some studies have reported that the thymus tissue or cultured thymic epithelial cells transplantated to immunodeficient mice, T cell function could be reconstructed; also studies have shown that removal of the thymus in mice occurrence of defects in immune function, easy to form tumors; after thymus transplantation, the anti-tumor capacity enhancement. The T cell function reconstruction and anti-tumor capacity enhancement must be based on the thymus transplant survival in the host and produce a variety of factors and cytokines in the thymus, and forming normal thymus micro-environment reconstruction. The cytokines that thymus producted could promot bone marrow stem cells differentiation into mature T lymphocytes. The mature T lymphocytes setted in peripheral immune organs and induced cellular immune activity, such as immune surveillance against tumors by killing tumor cytotoxicity cells. In recent years, hematological malignancies, severe refractory autoimmune disease, primary and secondary immunodeficiency diseases such as pose a great threat to human health, the re-establishment and restoration of normal immune function is an important part of curing these diseases and the ultimate goal. In this study, mice with congenital immune deficiency as the research object, established by transplanting human thymus tissue of human-mouse model, and then by transplanting human cord blood CD34+ cells of thymus transplantation and cell differentiation of CD34+ cells in the immune function of mice.Methods1. Human thymic tissue transplantation into nude mice:The nude mice were anesthetized, in turn cut back skin, muscle, fascia, exposing the left kidney, the thymus tissue implanted in the left kidney capsule, sutured. SPF fraction cages 60 d. End of the experiment, the anatomy of the thymus in mice observed in mice transplanted kidney survival, and by immunohistochemical detection of CD3 molecules and HLA-DR molecule expression in the transplanted thymus.2. Human cord blood CD34+ cells sorting:Human cord blood mononuclear cells were isolated and selected CD34+cells in healthy mothers natural childbirth points off, the umbilical cord blood after anticoagulation. The cord blood mononuclear cells (CBMC) were separated with centrifugation in lymphocyte separation medium,. CD34+cells were divided by immunomagnetic beads selected system(MACS). Couting living cells (95% above) by Trypan blue staining. Flow cytometry assay showed that CD34+cells accounted for 75%.3. nude mice umbilical cord blood CD34+ cells transplantation:Nude mice received the thymus transplantation 2 weeks later, the fresh separation umbilical cord blood CD34+cells were injected into nude mice by tail vein, each mouse input 1 x 106/mL CD34+cells 0.3 mL. The control group(non-thymus transplantation group) directly give CD34+cells transplantation. The experiment group and control group mice were breed continuely to 60 d.4. Immunity activities assays:The two groups nude mice were breed to 60d,10 mice of each group were detected T cells percentage and distribution in blood and peripheral immune organs. Human gastric cancer cell line BGC823 were transplanted to 5 mice of each group and tumor growth was observed everyday.5 The percentage of human T cells in nude mice vein blood:0.5 mL nude mice vein blood were taked from mice retinal. Human T cells and their subsets percentage were assayed with flow cytometry. After red blood cell lysis and PBS solution washed 3 times, FITC-anti-human CD3, FITC-anti-human CD4, FITC-anti-human CD8 monoclonal antibody and PE-anti-human CD25 monoclonal antibody were added respectely the vein blood and incubated for 30 min in 4℃. PBS washed 2 times. To the same antibody labeled the same as the control. Resuspended to 200μL cells detected by flow cytometry.6 The distribution of human CD3+cells in nude mice spleen:In order to detect the distribution of human CD3+T cells in experiment mice, the spleens were take out and fixed in the volume fraction of 10% neutral formalin. The spleens were paraffin embedded, sliced and stained with HE. The CD3 antigen expression in mice spleens were operated by immunohistochemical staining kit (SP method in Shan Beijing provided) according to specifications.7. The rejection of nude mice to xenograft tumor cells:Human gastric cancer cells BGC823 were cultured conventionally at 1×10'/mL cell concentration.0.2 mL BGC823 cell suspension were injected to nude mice subcutaneously at right shoulder. Tumor growth was observed everyday and the tumor size was acorded. Perpendicular to the maximum tumor diameter and 2 the product of the path (mm3) that the tumor size.8 Statistical analysis The statistical analysis of measurement data with SPSS 10.0 statistical software using mean±standard deviation (x±s) said, t test, count data usingχ2 test, P<0.05 for the difference statistically significant.Results1. Human thymic tissue under the renal capsule of nude mice survival and CD3, HLA-DR molecule expression:After 60d of human thymic tissue transplantation, the human thymic tissue survived in the renal capsule of nude mice. There were clear structure and diffuse expression of CD3 molecules and HLA-DR molecules in human thymic tissue under the nude renal capsule by HE staining and immunohistochemical staining.2 The distribution of CD3+ cells in nude mice spleen:After combine transplantation of thymic tissue and CD34+ cells, CD3+ T cells could be seen in experiment mice. CD3+ T cells were invisible in the group of umbilical cord blood CD34+cell transplantation.3 The percentage of human T cells in vein blood of experiment nude mice:CD3+ cells, CD4+cells, CD8+cells and CD4+CD25+cell percentage was significantly higher in the combine transplantation experimental groups than the control group of CD34+cord blood transplantation alone. P<0.054 The rejection of nude mice to xenograft tumor cells:The experimental groups of nude mice could resist tumor growth of human gastric cancer cell BGC823. The control group that CD34+cells alone transplantation could see tumor growth after BGC823 cells injected 3 days, and gradually growing, rapidly growing tumor mass.ConclusionThe thymus transplantation in nude mice can promote human umbilical cord blood hematopoietic stem/progenitor cells differentiate into functional T cell. The combine human thymus and CD34+ cell transplantation may be an important method of treatment of severe combined immunodeficiency disease and promote immune reconstitution.The human thymus-nude model can also be used as the mechanism of T cell differentiation. |
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