| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術研究所 === 95 === We previously demonstrated that Pichia pastoris-recombinant Ganoderma lucidum LZ-8 protein (rLZ-8) induces IL-2 expression via protein kinase-dependent signaling pathways. To continue our research, we were interested in the interaction of rLZ-8 and T cell surface membrane. We focused on three parts, i.e. T cell receptor (TCR) the proximal signalings, the signal transduction in the regulation of IL-2 secretion, and the putative rLZ-8 protein binding sites including CD45. Initially, we found that rLZ-8 dramatically induces phosphorylation of TCR-triggered proximal signaling molecules, and the increasing is in a time-dependent fashion. Next, in order to examine the elements involved TCR activation and outcome of IL-2 secretion, we used various TCR signaling-defected cell lines such as J.gamma-1 (PLCγ1-deficient), J.CaM1.6 (intracellular calcium response-deficient), J.RT3-T3.5 (lack αβ heterodimer) and J45.01 (CD45-deficient). In brief, rLZ-8 stimulation of IL-2 is in a time and dose–dependent fashion in wild Jurkat. However, IL-2 secretion is defected in the testing TCR signaling deficient Jurkat cells, especially J45.01. Moreover, we investigated the role of CD45 in the TCR-mediated regulation of IL-2 secretion. First, using a CD45 inhibitor, we demonstrated that CD45 inhibitor reduced IL-2 secretion in both human primary T cells and Jurkat T cells in a dose-dependent manner. Secondary, comparing rLZ-8 stimulated tyrosine phosphorylation profile between Jurkat and J45.01 cells, we found more phosphorylation in wild Jurkat than that in J45.01 cells. In addition, rLZ-8 rapidly increased phosphorylation of ZAP-70, LAT, PLCγ1 and PKCθ in wild Jurkat cells, but not in J45.01 cells. Our current finding provided evidence that rLZ-8 protein interacted with CD45 that allows CD45 to signal through the TCR-mediated signaling to produce IL-2 from human T cells, and free monosaccharides may reduce IL-2 expression due to decrease the interaction.
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