Studies On Murine Thymus And Thymus Function Restoration

Chapter 1 Establishment of accurate methods to evaluate murine thymus functionand statusAim: To evaluate the thymus function and status accurately, two methods should be developed. One is real-time PCR method to detect the new marker of naive T cell (sjT -RECs) levels in murine thymocytes and splenocytes.The amount of naive T cell and thymus function can be reflected by the method. The other is real-time RT-PCR method to study thymic-associated genes expression. Thymic microenvironment can be valued directly by the method. Methods:Real-time PCR steps: Genomic DNA extraction; PCR amplification; RAG2 PCR product purification; standard plasmid construction; PCR optimization;the standard curve was constructed and simples' sjTRECs were detected by real-time PCR.Real-time RT-PCR steps( for GAPDH, LM02, Foxn1, IL-7): RNA extraction; First-strand synthesis of cDNA; PCR amplification; PCR optimization; Real-time PCR reaction; Amplification curve output; Ct data obtain; Calculation. Results:Real-time PCR: After the optimized real-time PCR, the obtained standard curve had high reliability ( slope:-3.738, R2=0.998) and dissociation curve had single peak; TR -EC and RAG copies could be obtained by the standard curve;The content of sjTRECs could be calculated.Real-time RT-PCR: After the optimized real-time PCR,S-shaped amplification curve and single peak dissociation curve had been obtained .Different samples' Ct data could be exported; According 2-(?)Ct method, the ratio of gene expression could beaccurately calculated.Conclusions: The methods to evaluate thymus function and status by real-time PCRwere developed successfully.Chapter 2 Researches on murine thymus function and status at different ageAim: Age-associated changes occur in murine thymus function and status. To provide foundation for further research, the process should be investigated and compared systemically.Methods: Comparison of immune organ weight and index; Morphologic method(HE staining); the FACS analysis of CD3,CD4,CD8 subpopulations in murine blood; real-time PCR to examine the content of naive cell ; real-time RT-PCR to evaluate the expression of thymus- associated gene.Results: The weight and index of murine thymus and spleen decreased at 12w-old mouse; The HE staining results showed that thick thymus cortex ,dense cell and clear demarcation of cortex and medulla were morphologic characters of young mouse thymus.Old mice had opposite characters; The ratio of CD4 SP cell and CD8 SP was highest in 6w-old mouse, but began to reduce from 12w;The results of real-time PCR showed that naive cell always in thymus increased from new-born to mature stage(1-12w). But it failed from 12w till 36w.The naive T cell in peripheral was highest at lw,then declined; Compared with 1w-old mouse, the gene expression ratios of 36w-old mouse: LMO2:2.54±0.08,Foxnl:0.68±0.02,IL-7:0.15±0.03.The results of real-time RT-PCR indicated that old mouse thymic stromal cell avidity decreased. The production of T cell was blocked .As a result, the thymus microenvironment was destroyed.Conclusions: Tradition immunology index could reflect the degree and stability of immune system. The result of real-time PCR could reflect the detailed aged-associated changes of naive T cell. The result of real-time RT-PCR could evaluate the status in thymic microenvironment. All above methods could be validated each other. But real-time PCR was the most accurate method to evaluate the thymus function and status.Chapter 3 Development of immunosenescence study system and model in vitro andin vivoAim: To develop reliable man-made system and model and to find the way to control immunosenescence in vivo by using model in vitro, the effect of D-Gal on immunosen-scence at cellular, organic and organismal levels should be estimated. Methods: We developed the cellular system of D-Gal induced immunosenescence by ConA-induced proliferation of thymocytes and splenocytes and detected with 3H-TdR incorporations; we developed the organic system of D-Gal induced immunosenescence by young thymus organ culture system and detected with HE staining and PI flow cytometry analysis;we also developed the organismic model of D-Gal induced immun -osenescence by injection to mice and detected the model with real-time PCR to evaluate sjTRECs.Results: D-Gal had significantly decreased thymocytes and splenocytes proliferation ; After 15d organ culture, HE staining showed that control group thymus status was good and had no cell apoptosis phenomenon .In contrast, D-Gal treated group thymus cortex and medulla cell became sparse and in apoptosis ; After 3d, 7d, 11d, 15d organ culture, apoptosis percentage respectively was 9.19±0.41,21.65±2.68,28.59±0.93,46.36± 2.20(P<0.05);Compared with control group, mice treated with D-galactose in vivo showed the decreased content of naive T cell in both thymocytes and splenocytes. Conclusion: D-Gal could induce effectively immunosenescence in vivo and in vitro.Chapter 4 Investigation and detection of the potential and available methods tothymus function restoration and influences on immunosenescence Aim: To restore and regenerate thymus function, several potential and available methods should be established. The feasibility and mechanism of these methods should be investigated at the same time.Methods: Thymic stromal cell and bone stromal cell were cultured in vitro. We established the two-dimensional microenvironment using those cell compartment microenvironment. Combined with the man-made immunosenescence system in vitro (chapter 3), we investigated the two-dimensional microenvironment influence onimmunosenescence and thymus function. At organismic level, we transplanted young mouse pineal to old mouse thymus. Using the real-time PCR method, we evaluated the thymus function and status after pineal transplantation .Results: Compared with D-Gal control group, cell proliferation significantly increased in the co-culture with thymus stromal cell group. After thymus stromal cell transplant, there were increased significant content of naive T cell. Compared with D-Gal control group, cell proliferation had no significant change in the co-culture with bone stormal cell group. But after cell transplant ,there were still increased significant content of naive T cell.The pineal transplantation group had significant increased compared with control group.And gene expression ratio were LMO2:0.78±0.08,Foxn 1:5.27+ 1.58, IL7:3.46± 1.84,which indicated the thymus microenvironment might happen rebuilding. Conclusion: we established successfully several potential and available methods.