孔祥智Kung, Hsiang-Chih臺灣大學:免疫學研究所徐維璟Hsu, Wei-ChingWei-ChingHsu2010-05-102018-07-092010-05-102018-07-092009U0001-1708200918093800http://ntur.lib.ntu.edu.tw//handle/246246/181810T-cell receptor ζ chain-associated protein kinase 70 (Zap-70) is important for T cell development and activation. Both Zap-70-deficient humans and mice suffer from severe combined immunodeficiency (SCID) due to highly deficient T cell development. In Zap-70-knockout mice, intrathymic T cell developmental is blocked at the CD4+CD8+ double positive stage and no αβ-TCR+ T cells are seen. Here we made use of an ENU-induced Zap-70 mutant mouse, P358, characterized by a point mutation that results in a C563S amino acid change in the kinase domain. P358 mice are different from Zap-70-null mice in that significant numbers of CD4+ and CD8+ T cells develop. Zap-70 mRNA is expressed at similar levels in wildtype and P358 T cells, but Zap-70 protein expression in P358 is three- to four-fold reduced when compared to wildtype. Using TCR-stimulated P358 thymocytes or spleen cells, there is significant but reduced Lat phosphorylation. Functional studies reveal that P358 CD4+ T cell shows Th2 cytokines. In addition, the proliferation of P358 CD4+ and CD8+ T cells are defective upon stimulation by plate-bound anti-CD3. The P358 ENU mutant is therefore a Zap-70 hypomorphic model and has revealed new aspects of how the single C563S amino acid change affects protein turnover or function. This novel mouse mutant model is a tool that may help probe questions such as differential thresholds of Zap-70-dependent pleiotropic functions, mechanism of intrathymic selection processes, and how dysregulated immune cells and immune responses are generated.Abstract……………………………………………………………… iibstract (Chinese)…………………………………………………… iiihapter I Introduction …………………………………………….. 1 1.1 Background information and related studies…………………. 1hapter II Material and Methods…………………………………. 7 2.1 Mice…………………………………………………………... 7.2 Surface marker detection by flow cytometer………………….. 7.3 Intracellular staining of Zap-70……………………………….. 8.4 Real-time PCR………………………………………………… 9.5 IFN-γ, IL-5, IL-10, and IL-13 detection by ELISA…………… 10.6 IL-4/2 functional bioassay…………………………………….. 11.7 Spleen CD4+ and CD8+ T cell isolation by panning…………... 12.8 T cell activation………………………………………………... 13.9 293T cell transfection…………………………………………. 14.10 Immunoprecipitation…………………………………………. 15.11 Western blot………………………………………………….. 16hapter III Experimental Results………………………………… 18 3.1 Altered T cell development in P358 mice……………………... 18.2 The expression of Zap-70 shows post-transcriptional down-regulation in P358 mice………………………………… 18.3 Impaired kinase activity of P358 Zap-70 in 293T cell………... 20.4 Reduced phosphorylation of Lat in P358 thymocyte………….. 20.5 Both Zap-70 wt and Zap-70P358 can dimerization……………… 21.6 TCR-mediated activation of T cell is deficient in P358 mice. 21.7 P358 CD4+ T cells produce Th2 cytokines……………………. 22 3.8 Summary………………………………………………………. 23hapter IV Discussion………………………………………………. 24 4.1 Post-transcriptional down-regulation of Zap-70P358…………... 24.2 The insistent results of phosphorylated Lat in thymocyte ersus transfected 293 T cell………………………………….. 25.3 Dimerization of Zap-70……………………………………….. 27.4 P358 CD4+ T cell exhibits Th2-trended cytokine expression profiles………………………………………………………… 27eference…………………………………………………………….. 29hapter V Experimental Figures…………………………………… 37igures…………………………………………………………… 37application/pdf769581 bytesapplication/pdfen-USZap-70ENUT 細胞T cellhttp://ntur.lib.ntu.edu.tw/bitstream/246246/181810/1/ntu-98-R96449003-1.pdf