Mice Thymus And Its Response To Heterologous Antigen Cells

Back ground and objectiveThe thymus is a specialized primary lymphoid organ in the mouse, generating mature T lymphocytes to the peripheral circulation; it provides a rigid and complex microenvironment for T cell development and migration. Thymus stromal cells, soluble molecules and extracellular matrix compose the thymus microenvironment. Thymus stromal cells are mutually connected to form a network structure, the surfaces of these cells carry large amounts of surface molecules, and can secrete a variety of thymus hormones and cytokines, thus provide a stable microenvironment forthymocytes development and maturation. T cells in the thymus undergo TCR rearrangement, positive and negative selection and eventually develop into mature T cells and migrate to peripheral immune organs.According to the theory of central tolerance, peripheral tolerance on allogeneic antigens can be induced when T cells are exposed to the same antigen during development in the thymus. At the same time, blood-thymus barrier proved a stable microenvironment for T cell development, foreign macromolecular substances and antigens cannot entry the thymus easily, so the intrathymus injection of allogeneic antigen can escape immune surveillance. To better study the biological characteristics of heterogeneous antigen in the body and the body’s immune tolerance and immune rejection. We will injection6to8weeks of mice thymus lymphocytes and human liver cancer cells of Hep-G2to C57BL/6mice thymus that is10to15days, We use Hep-G2as the heterogeneous antigen from different tissues which has a large difference in MHC, as control, we also use PBS as the heterogeneous antigen which a little difference in MHC, so we can know the fate of heterogeneous antigen in thymus and the change of thymus microenvironment. By the methods of Impact on the cells number in the thymus and thymus weight after injection xenogeneic antigen to intrathymus, FCM analysis of mice thymus CD4CD8T cells changes, Analysis of tissue sections of after intrathymus injection of xenogeneic antigen, At the cellular level in the process of development of T cell differentiation, thymus microenvironment play to its function and T cells and thymus of the interaction between other types of cells are studied on the basis of further do thymus microenvironment, thymus injection of exogenous antigen (Hep-G2liver cancer cells) after the microenvironment of the electron microscope, the ultrastructural and molecular level development level of T cell differentiation process and the interaction between thymus microenvironment.Methods1. Transplantation of HEP-G2cells into mice thymus and histological analysis1) The development analysis of mice thymus;2) Cells cultured and Dil labeling;3) Simplification of intrathymus injection technique;4) Injection efficiency assessment:anatomic observation and FCM analysis;5) Mice weight change and injection technique assessment.6) Preparation of paraffin section and HE staining;2. Mice thymus responses to heterologous antigen cells after intrathymus injection of HEP-G2cells.1) Cell culture and Dil labeling;2) Transplantation of human hepatocellular carcinoma cells into mice thymus;3) Mice thymus weight and thymocytes number analysis;4) Peparation of paraffin section and HE staining;5) FCM analysis of cell subset proportion in mice thymus (T cell、NK/NKT cells and regulatory T cells);6) FCM analysis of thymus cell apoptosisResultsThe first partAnalysis on the development of mice thymusMice thymus developed rapidly before sex maturation, and reached the peak in adolescence, then it began to atrophy; mouse thymus size also grows along with the age, we select the first intercostal space as the injection area.Dil labeling and fluorescence observationMice thymus cells and human hepatoma cells were labeled successfully in the medium containing20μg/ml Dil after1hour, cells emit strong red fluorescence under fluorescence microscopy.Injection efficiency evaluationThrough the anatomical observation, we calculated the injection rate, it is91.67%in adult mice group, and86%in neonatal mice group; the results of FCM analysis confirmed that this method can ensure the accuracy of injection.Injection methods on mice injury severity assessmentThrough the analysis of mice weight and behavioral observation, we found their was no significant difference between the experimental group and the control group, we considered this injection method did little harm to mice, and worth application and promotion.Paraffin HE staining and observationHuman hepatocellular carcinoma cells in mice thymus were attacked and killed by lymphocytes, lymphocytes infiltration and macrophage recruitment was detected3days after injection; most of the tumor cells were dead and surrounded by dense lymphocytes7days after injection; there was no tumor cells with intact cellularity14days after injection, and a mass of lymphocytes were detected around tumor remains; no tumor cells could be detected21days after injection.The second partChanges of thymus weight and total cell numberCompared with the parallel control group, experimental mice thymus weight was significantly increased on days7,14and21after injection, while the total number of thymus was significantly reduced on the same time after injection (P<0.05).Injury of mice thymus caused by intrathymus injectionMechanical damage was detected3days after intrathymus injection of human hepatoma cells, some were caused by the injection needle, and others were the effects of injection needle combined with the thrust of HCC cells in the process of injection. Cortex/medulla ratio change of mice thymus Large area of medulla regions were detected around human tumor cells3days after injection, and medullary areas increased significantly, whereas the cortical area decreased14days after injection.Changes of thymus epithelial cellSpindle epithelial cells aggregated and surrounded human tumor cells after intrathymus injection, a mass of epithelial cells surrounded human tumor cell debris and formed elliptical structures16days after injection, stratified squamous epithelium surrounded human tumor cell debris and formed the concentric circles structure on days21and28after intrathymus injection; thymus tissue came back no normal2months after injection.Epithelial cell enrichment was detected in the area of thymus spaces after intrathymus injection, and it revealed that epithelial cells were also involved in thymes tissue reconstruction process.FCM analysis of cell subsets in mice thymusCompared to control group, percentage of CD4CD8double positive cells in experimental mice group decreased significantly, while CD4CD8double negative cells, CD4/CD8single positive cells and the percentage of NKT cells increased markedly1week after injection; percentage of regulation of T cell also increased2weeks after injection; there was no significant changes in the percentages of T lymphocyte, NK/NKT cell3weeks after injection.Thymocytes apoptosis analysisCompared to control group, percentage of thymocytes apoptosis in experimental group increased significantly1week after injection; and there was no significant changes in thymocytes apoptosis3weeks after injection.The third partAnalysis of tissue sections of after intrathymus injection of xenogeneic antigenHistological analysis found that the thymus early after injection, Hep-G2cell integrity, some undergoing mitosis, but as time goes on, Human liver cancer cells Hep-G2started to gather around a large number of lymphocytes, and the same time,21days arrived cannot detect the cancer cells.We can see a lot of squamous epithelial cells, which may be associated with the thymus tissue structure reconstruction. Human liver cancer cells Hep-G2in mice thymus ultimately did not escape the immune cells attack and death. The Hep-G2cells and the survival time of debris may for up to21days or so.Conclusions1. We simplified the intrathymus injection technique, and established a simple intrathymus injection technique for mice.2. We confirmed the fate of human hepatocellular carcinoma cells in mice thymus, and approved primarily that macrophages, NK/NKT cells and mature T cells were involved in the immune attack and removal of xenograft of human hepatocellular carcinoma cells.3. Intrathymus injection of human hepatocellular carcinoma cells resulted in certain pathological injury to mice thymus, including mechanical injury caused by injection, imbalance of cortex and medulla ratio, thymocytes number increase and epithelial cell hyperplasia, but these injuries disappeared and thymus returned to normal as tumor cells were killed and cleared up.4. T cell development and various types of cell subsets proportions were influenced by intrathymus injection of human hepatocellular carcinoma cells, thymocytes apoptosis increased, CD4CD8double positive cell percentage significantly decreased while CD4/CD8single positive cell, regulatory T cell and NK/NKT cell proportions increased, but when tumor cells were cleared, the percentages of various cell subpopulations returned to normal.