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Snk-SPAR途径在大鼠脾淋巴细胞中的初步研究

发布时间:2019-04-22 07:32
【摘要】: T淋巴细胞的活化是机体启动适应性免疫反应的关键事件,在T细胞发育阶段和成熟T细胞阶段,对T细胞受体(T-cell antigen receptor, TCR)表达水平的动态调节对T细胞应答起重要作用,进而对T细胞的发育、活化、存活、死亡起关键性作用。最近研究表明,TCR信号领先于免疫突触(immunological synapse ,IS)的形成,TCR在突触中央集聚成簇不但促进TCR信号传递,而且加强TCR下调和内化的几率,提出IS是一类自适应性控制器,在T细胞与特异性配体应答过程中调节TCR信号的强度和持续时间。Gascoigne亦证实某些T细胞信号领先于IS成熟之前,但T细胞的充分活化有赖于IS稳定持续的信号。因此对免疫突触调控的更好的理解将极大的推动TCR免疫识别和T细胞活化机制的认识。 血清诱导激酶(serum-inducible kinase,Snk)是丝氨酸/苏氨酸特异性马球样激酶(polo-like kinases,Plks)家族成员之一,是G1期Plk ;树突棘相关Rap-特异性GTPase-活化蛋白(spine- associated Rap guanosine triphosphatase activating protein ,SPAR)位于树突棘,SPAR以分子桥梁的形式与突触后表面蛋白,脚手架蛋白和肌动蛋白相连,是通过调节肌动蛋白重排控制树突棘形态的多域突触后蛋白。神经元活化能诱Snk表达,Snk靶向树突棘,与SPAR上的肌动蛋白调节域相结合使SPAR发生磷酸化,泛素连接酶识别磷酸化SPAR后,SPAR即被泛素修饰,随后SPAR经蛋白酶体途径发生降解,造成PSD95等脚手架蛋白的丢失,引起树突棘形态学变化即由钝圆形变成狭长状,这就形成了Snk触发泛素化并进而降解SPAR的Snk-SPAR途径。Snk-SPAR途径可能对突触兴奋性提高后的突触功能缓冲起稳态调节作用,该途径能维持局部突触界面修饰为基础的突触可塑化的稳定性,能全面调整神经元活化水平,对Snk-SPAR途径的调控将成为控制突触重构的强有力的方法。 尽管神经突触和免疫突触显著不同,但二者共享许多相似的特性,神经系统和免疫系统通过突触在构成它们的两个细胞之间直接传递和转换强大的分泌控制信号,且这种刺激信号是特异的和保守的;结构上神经突触和免疫突触都由中央活化区和富含粘附分子的周边区组成,中央区具有分泌功能,周边区具有内吞作用。至今为止神经生物学家和免疫学家已发现有些分子为两者所共有。因此设想在免疫突触中或许也存在Snk-SPAR途径调控免疫突触重构进而影响免疫活化网络的可塑性。本文是该设想的第一步探索-即探讨Snk-SPAR途径是否存在于免疫系统。 本研究选取健康成年SD大鼠(体重为275±25 g,雌雄各半)为研究对象,常规制备大鼠脾淋巴细胞悬液,调细胞浓度至2×106/ml。首先用MTT法确定刀豆蛋白A(ConcanavalinA,Con A)激活淋巴细胞的最适浓度之后,将Con A干预的脾淋巴细胞设为7个不同的培养时间点(设空白对照组),分别收集各时间点淋巴细胞,提取总RNA,用RT-PCR法初步检测Snk、SPAR mRNA的动态表达情况(同时设大鼠海马组织为阳性对照)。在此基础上,制备脾CD4+T细胞,将Con A干预的CD4+T细胞设为0.5 h、1 h两个培养时间点(设空白对照组),分别收集各时间点CD4+T细胞,提取总蛋白,用Dot blot法进一步在蛋白质水平上验证Snk、SPAR存在于淋巴细胞中。 实验结果: 1.确定刀豆蛋白A的最适活化浓度MTT法确定Con A活化淋巴细胞的最适剂量为5μg/ml。 2. Snk mRNA在脾淋巴细胞中的动态表达 未加Con A的10 min对照组,脾淋巴细胞中存在Snk mRNA的基础性表达;用Con A活化的7个时间点,Snk mRNA表达呈动态变化:培养10 min时,Snk条带清晰,表达高于对照组(P0.05),培养达到0.5 h时,Snk表达升高且达到高峰(与其他各组比较均具有统计学差异,P0.05),培养1 h, Snk表达减少,当培养2 h时, Snk表达继续下降,培养6 h、24 h、72 h时Snk表达恢复到基础水平,与10 min对照组比较已无明显差异(P0.05)。 3. SPAR mRNA在脾淋巴细胞中的动态表达 不加Con A的10 min对照组淋巴细胞中存在SPAR mRNA的基础性表达;在Con A活化的7个时间点中,SPAR mRNA表达呈动态改变:培养10 min时,SPAR条带清晰(与对照组无差异,P0.05),培养0.5 h时,SPAR mRNA较10 min时表达减少(P0.05),培养1 h时,SPAR mRNA的表达较0.5 h进一步下降并降至低谷,当培养达到2 h时, SPAR mRNA较1 h表达无差异(P0.05),随着培养时间的进一步延长,SPAR mRNA表达上升,培养6 h、24 h、72 h时SPAR mRNA表达呈持续增加态势。 4. Snk、SPAR蛋白在脾CD4+T细胞中的表达 不加Con A的0.5 h对照组CD4+T中存在Snk、SPAR蛋白的基础性表达;在Con A活化的0.5 h、1 h时间点,检测到CD4+T中存在Snk蛋白的表达, SPAR蛋白在0.5 h时有表达,在1 h未检测到。 结论: 1.正常大鼠脾淋巴细胞存在Snk mRNA的基础表达。受Con A激活时Snk mRNA表达增加,且该表达在激活早期呈先升后渐趋恢复到基础水平的动态变化的过程。 2.正常大鼠脾淋巴细胞中存在SPAR mRNA的基础性表达;在Con A活化的7个时间点中,SPAR mRNA表达呈先从基础水平开始减少,6 h后又渐呈持续增加态势。 3.正常大鼠脾CD4+T细胞中存在Snk、SPAR蛋白的表达
[Abstract]:The activation of T-lymphocytes is a key event in the body-initiated adaptive immune response, and the dynamic regulation of T-cell receptor (TCR) expression level plays an important role in the T-cell response and the development of T-cells in the T-cell stage and the mature T-cell stage. Activation, survival, and death play a critical role. Recent studies have shown that TCR signals lead to the formation of immune synapses (IS), and TCR is clustered in the center of the synapse to not only promote TCR signal transmission, but also increase the probability of down-regulation and internalization of TCR. It is suggested that IS is a kind of self-adaptive controller. The intensity and duration of the TCR signal are adjusted in the T cell and the specific ligand response. Gascoigne also confirmed that some T-cell signals were ahead of the IS maturation, but the full activation of T-cells depended on an IS-stable continuous signal. Therefore, the better understanding of the regulation of immune synapse will greatly promote the recognition of TCR immune recognition and the mechanism of T-cell activation. The serum-induced kinase (Snk) is one of the members of the serine/ threonine-specific horse-like kinases (Pks) family, which is the G1 phase Plk; a dendritic spine-related Rap-specific GTPase-activated protein, S the PAR) is located in the dendritic spine, and the SPAR is linked to the postsynaptic surface protein, the scaffold protein and the actin in the form of a molecular bridge, The activation energy of the neuron induces the expression of Snk, the Snk is targeted to the dendritic spine, and the SPAR is phosphorylated by the combination of the actin regulation domain on the SPAR. After the ubiquitin ligase recognizes the phosphorylated SPAR, the SPAR is modified by the ubiquitin, and then the SPAR is degraded by the proteasome pathway, resulting in the PSD95. The loss of the scaffold protein causes the morphological change of the dendritic spine to become long and narrow by the blunt circle, which forms the S that the Snk triggers the ubiquitination and further degrades the SPAR. The Nk-SPAR pathway can regulate the synaptic function after synaptic excitability, which can maintain the stability of the synaptic plasticity on the basis of local synaptic interface modification. The regulation of the pathway of Snk-SPAR will be the control of the synapse. Although the synapses and the immune synapses are significantly different, they share a lot of similar characteristics, and the nervous system and the immune system pass through the synapses directly to transfer and transform a powerful secretion control between the two cells that form them. the system consists of a central activation region and a peripheral region rich in adhesion molecules, The central region has the function of secretion, and the peripheral region has endocytosis. So far, the neurobiology Family and immunologists have found that some molecules are common to both. It is therefore envisaged that there may also be Snik-SPAR pathway regulation in the immune synapse In this paper, the first step in this paper is to probe into the effect of the presynaptic reconstruction of the phytophthora, which can affect the plasticity of the immune-activated network. Snik-SPAR pathway is present in the immune system. Healthy adult SD rats (275 to 25 g, and half of male and female) are selected as the study subjects. In the conventional method, the spleen lymphocyte suspension of the rat was prepared, and the concentration of the cell was adjusted to 2-106/ ml. After the optimal concentration of the activated lymphocytes of the concanavaline A (Con A) was determined by the MTT method, the splenocytes of the Con A were set to 7 different culture time points (with the blank control group), and the cells were collected separately. The detection of Snk, SP by RT-PCR was carried out by RT-PCR. The expression of AR mRNA was the positive control of the rat's hippocampus. On the basis of this, the spleen CD4 + T cells were prepared, and the CD4 + T cells of Con A were set to 0.5 h and 1 h. The CD4 + T cells were collected at each time point. Take total protein, and use Dot blot the method further At the protein level, Snk and SPAR were present in the lymphocytes. The results of the experiment were as follows: 1. Determination of Stool-bean The optimal activation concentration of the protein A was 5. m u.g/ ml.2. The dynamic expression of Snk mRNA in the spleen lymphocytes was not observed in the control group, the spleen and the lymph nodes without Con A. At 7 time points activated by Con A, the expression of Snk mRNA was dynamic change: at 10 min, the expression of Snk was clear and the expression was higher than that of the control group (P0.05). When the culture reached 0.5 h, the expression of Snk increased and reached the peak. (There was a statistical difference with the other groups, P0.05). The expression of Snk decreased when cultured for 1 h, and when cultured for 2 h, Snk The expression of SPAR mRNA in the spleen lymphocytes was not significant (P0.05). The expression of SPAR mRNA in the 10-min control group with Con A was the basic expression of SPAR mRNA. The expression of SPAR mRNA in 7 time points activated by Con A showed a dynamic change: when cultured for 10 min, the SPAR band was clear (no difference with the control group, P0.05). At the time of incubation for 0.5 h, the expression of SPAR mRNA was decreased at 10 min. (P0.05), when cultured for 1 hour, the expression of SPAR mRNA was 0.5 h. One step is down and down to the valley, and when the culture reaches 2 h, the SPAR mR There was no difference in the expression of SPAR (P0.05). The expression of SPAR mRNA was increased with the increase of the culture time, and the expression of SPAR mRNA in cultured for 6 h,24 h and 72 h. R protein in spleen CD4 +T缁,

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