The Research Of SNK-SPAR Expression In Post-Immunological Synapse CD4~+T Lymphocyte

Backgroud and objectives:Immunological synapse is a special signal transduction area locating between T cell and antigen presenting cell (APC) contact surface, which can form stable contact and signal transduction between T cell and accessory molecules. Some studies indicate that T cell activation following immunological synapse cleft ubiquitination process. Immunological synapse is a not only triggered site of activation process, but also controlling whole activation process with signal transducers. A important characteristic of T cell recognize antigen is that TCR peptide-MHC interaction is a self-limited course, but at present we do not know TCR acceptor mediated cellular localization responsive ubiquitination mechanism.Now we know that protein ubiquitination can help protein change synapse function at nerve ending. Neuron activation dependent serum inducible kinase (SNK)-spine-associated Rap guanosine triphosphatase (GTPase) activating protein (SPAR)-ubiquitin proteasomes pathway is a poikilostasis effect for synapse function amortization of synapse excitability elevation. This pathway can maintain synapse plastic stability basing local synaptic interface modification, which can overall regulate neuron activation level. Regulating this pathway will become a drastic way of controlling synapse reconstruction.Nervous synapse and immunological synapse share much similar characteristics. The nervous system and immune system can directly convey and transduce powerful superior position secretory control signals through synapse. Nervous synapse and immunological synapse are both built around a microdomain structure comprising central active zones of exocytosis and endocytosis encircled by adhesion domains. So we presume that SNK-SPAR pathway probably participate in post-immunological synapse T cell activated self-limited regulation through controlling immunological synapse intensity. This article carried out earlier basal demonstration for presume. We observed that SNK/SPAR expression in post-immunological synapse CD4~+T cell and dynamic state distribution process in RNA and protein level, which probably revealed significance about SNK-SPAR pathway in immunological synapse, and provided essential preparatory work for searching special intervention latent target which could make T cell response to recover homeostasis.Methods:Preparing purified rat spleen CD4~+T lymphocyte with nilon bristles column and indirect Panning way, then used optimum dose 5μg/ml Con A to active CD4~+T lymphocyte, which are divided into seven groups and control groups according to cultivating time (10min,30min,1h,2h,6h, 24h,48h). We detect expression change of SNK-SPAR mRNA and protein level in the CD4~+T lymphocyte by RT-PCR and immunofluorescence.Results:1. SNK mRNA of experimental groups are expressed at seven time points, whose expression peak is at 30 minute, which is statistically significant comparing to 10min, 2h, 6h, 24h and 48h(P<0.05). SNK mRNA expression is least at 48h, but strap is clear. SNK mRNA of control groups are expressed from 10min to 24h, whose expression peak is also at 30 minute which is statistically significant comparing to 2h, 6h and 24h (P<0.05), and no expression at 48h.SPAR mRNA of experimental groups are expressed at seven time points, whose least expression is at 48h, which is statistically significant comparing to 6h, 24h(P<0.05). Changes of control groups are similar to experimental groups, whose least expression is at 48h, which is statistically significant comparing to 10min, 30min, 1h, 2h and 6h(P<0.05).SNK mRNA expression peak of experimental groups is not statistically significant comparing to control groups, which is statistically significant at time point elongation comparing to 48h (P<0.05). SPAR mRNA of experimental groups have significant difference comparing to control groups at 24h and 48h (P<0.05). SNK -SPAR mRNA expression level are not obviously related in the CD4~+T lymphocyte.2. SNK protein expression peak of experimental groups and control groups are both at 1h, then quickly decreasing, which are significant deviation comparing to 10min, 30min, 2h, 6h, 24h and 48h (P<0.05).SPAR protein expression of experimental groups is least at 48h, which is not significant deviation comparing to other time points (P>0.05). SPAR protein expression of control groups is also least at 48h, which is statistically significant comparing to 10min, 30min, 1h and 2h (P<0.05).SNK protein of experimental groups have significant difference comparing to control groups at 2h,24h and 48h (P<0.05). SPAR protein of experimental groups have significant difference comparing to control groups at 48h (P<0.05). SNK -SPAR protein expression level are not obviously related in the CD4~+T lymphocyte.Conclusions:1. SNK/SPRA mRNA and protein are surely expressed in CD4~+T cell.2. SNK-SPRA pathway regulation is insufficient in CD4~+T cell culture which is activated by Con A.