【摘要】： T淋巴細(xì)胞的活化是機(jī)體啟動(dòng)適應(yīng)性免疫反應(yīng)的關(guān)鍵事件,在T細(xì)胞發(fā)育階段和成熟T細(xì)胞階段,對(duì)T細(xì)胞受體(T-cell antigen receptor, TCR)表達(dá)水平的動(dòng)態(tài)調(diào)節(jié)對(duì)T細(xì)胞應(yīng)答起重要作用,進(jìn)而對(duì)T細(xì)胞的發(fā)育、活化、存活、死亡起關(guān)鍵性作用。最近研究表明,TCR信號(hào)領(lǐng)先于免疫突觸(immunological synapse ,IS)的形成,TCR在突觸中央集聚成簇不但促進(jìn)TCR信號(hào)傳遞,而且加強(qiáng)TCR下調(diào)和內(nèi)化的幾率,提出IS是一類自適應(yīng)性控制器,在T細(xì)胞與特異性配體應(yīng)答過程中調(diào)節(jié)TCR信號(hào)的強(qiáng)度和持續(xù)時(shí)間。Gascoigne亦證實(shí)某些T細(xì)胞信號(hào)領(lǐng)先于IS成熟之前,但T細(xì)胞的充分活化有賴于IS穩(wěn)定持續(xù)的信號(hào)。因此對(duì)免疫突觸調(diào)控的更好的理解將極大的推動(dòng)TCR免疫識(shí)別和T細(xì)胞活化機(jī)制的認(rèn)識(shí)。 血清誘導(dǎo)激酶(serum-inducible kinase,Snk)是絲氨酸/蘇氨酸特異性馬球樣激酶(polo-like kinases,Plks)家族成員之一,是G1期Plk ;樹突棘相關(guān)Rap-特異性GTPase-活化蛋白(spine- associated Rap guanosine triphosphatase activating protein ,SPAR)位于樹突棘,SPAR以分子橋梁的形式與突觸后表面蛋白,腳手架蛋白和肌動(dòng)蛋白相連,是通過調(diào)節(jié)肌動(dòng)蛋白重排控制樹突棘形態(tài)的多域突觸后蛋白。神經(jīng)元活化能誘Snk表達(dá),Snk靶向樹突棘,與SPAR上的肌動(dòng)蛋白調(diào)節(jié)域相結(jié)合使SPAR發(fā)生磷酸化,泛素連接酶識(shí)別磷酸化SPAR后,SPAR即被泛素修飾,隨后SPAR經(jīng)蛋白酶體途徑發(fā)生降解,造成PSD95等腳手架蛋白的丟失,引起樹突棘形態(tài)學(xué)變化即由鈍圓形變成狹長狀,這就形成了Snk觸發(fā)泛素化并進(jìn)而降解SPAR的Snk-SPAR途徑。Snk-SPAR途徑可能對(duì)突觸興奮性提高后的突觸功能緩沖起穩(wěn)態(tài)調(diào)節(jié)作用,該途徑能維持局部突觸界面修飾為基礎(chǔ)的突觸可塑化的穩(wěn)定性,能全面調(diào)整神經(jīng)元活化水平,對(duì)Snk-SPAR途徑的調(diào)控將成為控制突觸重構(gòu)的強(qiáng)有力的方法。 盡管神經(jīng)突觸和免疫突觸顯著不同,但二者共享許多相似的特性,神經(jīng)系統(tǒng)和免疫系統(tǒng)通過突觸在構(gòu)成它們的兩個(gè)細(xì)胞之間直接傳遞和轉(zhuǎn)換強(qiáng)大的分泌控制信號(hào),且這種刺激信號(hào)是特異的和保守的;結(jié)構(gòu)上神經(jīng)突觸和免疫突觸都由中央活化區(qū)和富含粘附分子的周邊區(qū)組成,中央?yún)^(qū)具有分泌功能,周邊區(qū)具有內(nèi)吞作用。至今為止神經(jīng)生物學(xué)家和免疫學(xué)家已發(fā)現(xiàn)有些分子為兩者所共有。因此設(shè)想在免疫突觸中或許也存在Snk-SPAR途徑調(diào)控免疫突觸重構(gòu)進(jìn)而影響免疫活化網(wǎng)絡(luò)的可塑性。本文是該設(shè)想的第一步探索-即探討Snk-SPAR途徑是否存在于免疫系統(tǒng)。 本研究選取健康成年SD大鼠(體重為275±25 g,雌雄各半)為研究對(duì)象,常規(guī)制備大鼠脾淋巴細(xì)胞懸液,調(diào)細(xì)胞濃度至2×106/ml。首先用MTT法確定刀豆蛋白A(ConcanavalinA,Con A)激活淋巴細(xì)胞的最適濃度之后,將Con A干預(yù)的脾淋巴細(xì)胞設(shè)為7個(gè)不同的培養(yǎng)時(shí)間點(diǎn)(設(shè)空白對(duì)照組),分別收集各時(shí)間點(diǎn)淋巴細(xì)胞,提取總RNA,用RT-PCR法初步檢測(cè)Snk、SPAR mRNA的動(dòng)態(tài)表達(dá)情況(同時(shí)設(shè)大鼠海馬組織為陽性對(duì)照)。在此基礎(chǔ)上,制備脾CD4+T細(xì)胞,將Con A干預(yù)的CD4+T細(xì)胞設(shè)為0.5 h、1 h兩個(gè)培養(yǎng)時(shí)間點(diǎn)(設(shè)空白對(duì)照組),分別收集各時(shí)間點(diǎn)CD4+T細(xì)胞,提取總蛋白,用Dot blot法進(jìn)一步在蛋白質(zhì)水平上驗(yàn)證Snk、SPAR存在于淋巴細(xì)胞中。 實(shí)驗(yàn)結(jié)果: 1.確定刀豆蛋白A的最適活化濃度MTT法確定Con A活化淋巴細(xì)胞的最適劑量為5μg/ml。 2. Snk mRNA在脾淋巴細(xì)胞中的動(dòng)態(tài)表達(dá) 未加Con A的10 min對(duì)照組,脾淋巴細(xì)胞中存在Snk mRNA的基礎(chǔ)性表達(dá);用Con A活化的7個(gè)時(shí)間點(diǎn),Snk mRNA表達(dá)呈動(dòng)態(tài)變化:培養(yǎng)10 min時(shí),Snk條帶清晰,表達(dá)高于對(duì)照組(P0.05),培養(yǎng)達(dá)到0.5 h時(shí),Snk表達(dá)升高且達(dá)到高峰(與其他各組比較均具有統(tǒng)計(jì)學(xué)差異,P0.05),培養(yǎng)1 h, Snk表達(dá)減少,當(dāng)培養(yǎng)2 h時(shí), Snk表達(dá)繼續(xù)下降,培養(yǎng)6 h、24 h、72 h時(shí)Snk表達(dá)恢復(fù)到基礎(chǔ)水平,與10 min對(duì)照組比較已無明顯差異(P0.05)。 3. SPAR mRNA在脾淋巴細(xì)胞中的動(dòng)態(tài)表達(dá) 不加Con A的10 min對(duì)照組淋巴細(xì)胞中存在SPAR mRNA的基礎(chǔ)性表達(dá);在Con A活化的7個(gè)時(shí)間點(diǎn)中,SPAR mRNA表達(dá)呈動(dòng)態(tài)改變:培養(yǎng)10 min時(shí),SPAR條帶清晰(與對(duì)照組無差異,P0.05),培養(yǎng)0.5 h時(shí),SPAR mRNA較10 min時(shí)表達(dá)減少(P0.05),培養(yǎng)1 h時(shí),SPAR mRNA的表達(dá)較0.5 h進(jìn)一步下降并降至低谷,當(dāng)培養(yǎng)達(dá)到2 h時(shí), SPAR mRNA較1 h表達(dá)無差異(P0.05),隨著培養(yǎng)時(shí)間的進(jìn)一步延長,SPAR mRNA表達(dá)上升,培養(yǎng)6 h、24 h、72 h時(shí)SPAR mRNA表達(dá)呈持續(xù)增加態(tài)勢(shì)。 4. Snk、SPAR蛋白在脾CD4+T細(xì)胞中的表達(dá) 不加Con A的0.5 h對(duì)照組CD4+T中存在Snk、SPAR蛋白的基礎(chǔ)性表達(dá);在Con A活化的0.5 h、1 h時(shí)間點(diǎn),檢測(cè)到CD4+T中存在Snk蛋白的表達(dá), SPAR蛋白在0.5 h時(shí)有表達(dá),在1 h未檢測(cè)到。 結(jié)論: 1.正常大鼠脾淋巴細(xì)胞存在Snk mRNA的基礎(chǔ)表達(dá)。受Con A激活時(shí)Snk mRNA表達(dá)增加,且該表達(dá)在激活早期呈先升后漸趨恢復(fù)到基礎(chǔ)水平的動(dòng)態(tài)變化的過程。 2.正常大鼠脾淋巴細(xì)胞中存在SPAR mRNA的基礎(chǔ)性表達(dá);在Con A活化的7個(gè)時(shí)間點(diǎn)中,SPAR mRNA表達(dá)呈先從基礎(chǔ)水平開始減少,6 h后又漸呈持續(xù)增加態(tài)勢(shì)。 3.正常大鼠脾CD4+T細(xì)胞中存在Snk、SPAR蛋白的表達(dá)
[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緇，

本文編號(hào)：2462626