The Initial Study Of Snk-SPAR Pathway In Rat Splenic Lymphocytes

The activation of T lymphocyte is the critical event as body triggering adaptive immunity reaction. Dynamic regulating the expression level of the T cell antigen receptor (TCR) plays an important role in T cell immune response during both T cell developmental stage and mature T cell stage, and then is very critical for T cell development,activation,survival and death. Recent studies indicate that TCR signal emerging is ahead of the immunological synapse (IS) formation and TCR clustering in the center of IS not only facilitates TCR signal transmission but also enhances probability of TCR down regulation and internalization, therefore Their finding implies that IS is a self-adaptation controller, which modulates TCR signal intensity and duration in the process of immune response between T cell and specific ligand. Gascoigne also confirmed certain T cell signals is occurred before IS maturation, however, the sufficient activation of T cell is relied on the stable and durative IS signals. Accordingly, how to regulate IS is avail of understanding the mechanism of TCR immunological recognition and T lymphocyte activation.Serum-inducible kinase(Snk)belongs to a family of serine/threonine–specific polo-like kinases that are involved in cell cycle control. Snk expression is detected only in G1;Spine- associated Rap guanosine triphosphatase activating protein(SPAR)located in the dendritic spine, is a multidomain postsynaptic protein that controls dendritic spine shape by regulating actin arrangemen. SPAR associates with other postsynaptic surface and scaffolding proteins as well as with the actin cytoskeleton as style of molecule brige. Snk was induced in hippocampal neurons by synaptic activity and was targeted to dendritic spines. Snk bound to SPAR by actin regulatory domains and phosphorylated it.Aubiquitin ligase recognizes phosphorylated SPAR and modifies it with ubiquitin. SPAR is then degraded via the proteasome pathway. The dendritic spine loses the scaffolding protein PSD95 and changes its shape to become a long, thin protrusion as causes loss of mature dendritic spines. Snk could phosphorylate and activate a ubiquitin ligase that mediates the ubiquitination of SPAR. This is the Snk-SPAR signaling pathway which contributes to the mechanisms of global synaptic homeostasis and presents an attractive homeostatic mechanism for stabilizing the excitability of neurons homeostatic regulation is thought to occur through multiplicative changes in the strength and shape of all (or most) synapses. All of this imply modulating Snk-SPAR pathway is critical for synapse remodel.There are a number of parallel specificities between immunological synapse and neuron synapse by comparing them. The nervous system and immune system utilize synapses to directly convey and transduce highly controlled secretory signals between their constituent cell populations as well as the signals are specific and conservative. Each of these synaptic types is built around a microdomain structure comprising central active zones of exocytosis and endocytosis encircled by adhesion domains. Up to now, neurobiologist and immunologist have discovered that there are some molecules between of them. In light of this, we presumed that the Snk-SPAR pathway is occurred in IS which contributes to modulate IS reconstitution and influence plasticity of immune network. This text is the preliminary research of this idea, namely investigate whether the Snk-SPAR pathway is occurred in immune system.The experiment object is healthy and adult Sprague-Dawley(SD)rat whose weight is 275±25 g as well as female and male is half. Routine prepare rat splenic lymphocyte suspension, adjust cell concentration to 2×106/ml. First of all, apply MTT method to determine the optimal concentration of the ConcanavalinA(Con A)which is used for activating lymphocytic.Then culture the rat splenic lymphocytes which are treated with Con A according to seven different culture time poin(twithout Con A is blank control group), following collect each time point rat splenic lymphocytes, extract the total RNA , investigate dynamic expression of the Snk,SPAR mRNA through RT-PCR method(positive control is rat hippocampus tissue ). Prepare ratsplenic CD4+T cell, and then culture the rat splenic CD4+T cells which are treated with Con A(without Con A is blank control group);After cultured 0.5 h,1 h, collect each time point rat splenic CD4+T cell, extract the total protein. Apply Dot blot method to confirm that Snk,SPAR are existed in rat splenic CD4+T cells at the protein level.Results:1. determine the optimal activation concentration of the Con A The optimal activation concentration of the Con A through MTT method is 5μg/ml.2. Dynamic expression of the Snk mRNA in rat splenic lymphocyteIn the case of blank control group(without Con A, culture 10 min ),there is foundational expression of Snk mRNA in rat splenic lymphocyte; when culture 10 min, the level of Snk mRNA is higher than blank control group(P<0.05),while culture 0.5 h, the level of Snk mRNA is enhanced and up to the peak(compared with other each group, P<0.05), the level of Snk mRNA is reduced when culture 1 h and continus reduced after culture 2 h.While culture 6 h,24 h,72 h, the level of Snk mRNA is restored the basal level, there is no significant difference compared with blank control group(P>0.05)3. Dynamic expression of the SPAR mRNA in rat splenic lymphocyteIn the case of blank control group(without Con A, culture 10 min ),there is foundational expression of SPAR mRNA in rat splenic lymphocyte; The level of SPAR mRNA is changed dynamically during following seven time points(treated with Con A): when culture 10 minutes, there is no significant difference compared with control group(P<0.05),at the 0.5 h time point,the level of SPAR mRNA is lower than 10 min time point(P<0.05),while culture 1 h, the level of SPAR mRNA is continued decreased and up to the nadir, after culture 2 h, there is no significant difference between the 1 h and 2 h time poin(tP>0.05).as the culture time future extended, the level of SPAR mRNA is persistent advanced during 6 h,24 h,72 h time point.4. The expression of the Snk,SPAR protein in rat CD4+T cell.In the case of blank control group(without Con A, culture 0.5 h ),there are basal expression of Snk,SPAR protein in rat CD4+T cells; At the 0.5 h,1 h time point, Snk protein are detected in rat CD4+T cells which are treatedwith Con A; SPAR protein can be detected after cultured 0.5 h, however , at the 1 h time point, SPAR protein can not be detected.Conclusions:1. There is basal expression of Snk mRNA in normal rat splenic lymphocyte. The level of Snk mRNA is enhanced as the rat splenic lymphocyte is treated with Con A and the expression of Snk mRNA is changed dynamically ?2. There is basal expression of SPAR mRNA in normal rat splenic lymphocyte. As the rat splenic lymphocyte is treated with Con A, the level of SPAR mRNA is reduced from the basal level and increased persistently after cultured 6 h during the seven time points.3. There are expression of Snk,SPAR protein in normal rat splenic CD4+T cell.