SSeCKS在炎症活化星形胶质细胞中作用的初步研究

发布时间：2018-05-12 23:20

本文选题：SSeCKS + 蛋白激酶C　；参考：《南通大学》2008年硕士论文

【摘要】： [研究背景及目的] 中枢神经系统(Central nervous system,CNS)内炎症病理过程是以胶质细胞的活化和周围炎症细胞的浸润为主要特征的。作为CNS中数量最多的细胞类型,星形胶质细胞在维持中枢神经系统的稳态方面发挥重要的作用。当机体遭受外来炎症因子或微生物刺激时,星形胶质细胞被迅速激活并产生大量的炎性因子,这些细胞因子具有双向的调节作用:一方面,它们可以诱导更大量炎症因子在中枢神经系统内的产生,例如趋化因子、其它细胞因子、黏附分子等,并由此调节机体对外来刺激的免疫应答过程;另一方面,这些生物活性因子的过度释放会导致反应性胶质增生以及中枢神经系统内环境的破坏,阻碍轴突的再生,影响神经元的活性。因此,对炎症反应的适当调节对于中枢神经系统在免疫应答过程中稳态的维持具有重要的意义。研究表明,多种信号调节机制参与调节星形胶质细胞的活化过程。近几年的研究指出,蛋白激酶C(protein kinase C , PKC)信号途径在星形胶质细胞的激活过程中发挥重要作用,并参与调节星形胶质细胞分化、迁移以及细胞因子的释放等过程。而这种调节作用的发挥主要是依赖于PKC底物的锚定作用对其空间结构以及亚细胞定位的改变。我们所说的AKAP(A Kinase Anchoring Protein)家族的蛋白分子以及蛋白激酶C锚定蛋白就是这种具有锚定功能的蛋白,它们可以作为一种连接蛋白与激酶相结合并将其引导至胞内确定的结构上。本课题的研究对象SSeCKS就是锚定蛋白的一种,该分子最初被认为是有丝分裂的负性调节分子,是PKC的重要底物之一。作为AKAP蛋白家族的一员,SSeCKS的主要效应结构域可竞争性结合纤维状肌动蛋白(F-actin),钙调蛋白(calmodulin,CaM),细胞周期蛋白D(cyclinD),羧基端有蛋白激酶A(protein kinase A,PKA)的结合结构域。因此,SSeCKS可以通过选择性结合一些信号蛋白,整合细胞内信号。Lin等人的研究表明,PKCα可磷酸化SSeCKS并影响其细胞内定位;Kitamura等人发现,在炎症反应过程中,SSeCKS在肺、心脏、肾脏、脑组织中的表达明显增高,故认为SSeCKS是一种炎症反应蛋白;Sae-Won Lee等研究表明,SSeCKS在星形胶质细胞中有表达,并在血脑屏障功能的成熟和维护中发挥重要作用。这些证据提示SSeCKS可能与炎症过程中PKC介导的胶质细胞的活化有关。本研究主要对腹腔注射LPS后SSeCKS在脑组织的表达、定位情况进行了分析,并探讨了该分子在炎症活化星形胶质细胞过程中的作用与相关机制,为中枢神经系统炎症的治疗提供了新的思路。 [方法] 1.建立LPS致伤大鼠炎症模型。 2.运用Real-time PCR、Western blot的方法检测脑组织中SSeCKS表达的时空变化,运用免疫荧光双标,观察SSeCKS在脑组织的细胞定位;分析星形胶质细胞活化与SSeCKS表达水平之间的相关性。 3.分离培养并纯化大鼠星形胶质细胞。在细胞水平,运用Real-time PCR、Western blot、激光共聚焦等技术,检测LPS对SSeCKS表达、亚细胞定位的影响;分析炎症刺激,SSeCKS的表达水平以及PKC活性三者之间的相关性。 4.运用分子生物学技术构建SSeCKS RNA干扰质粒,转染细胞。运用免疫细胞荧光,Western blot,ELISA,流式细胞仪(Fluorescein activated cell sorter,FACS),免疫沉淀等方法分析干扰SSeCKS表达对活化过程中星形胶质细胞生物合成以及增殖水平的影响以及相关的信号传递机制。 [结果] 1. 1)LPS致伤大鼠后早期, SSeCKS在脑组织中反应性表达上调,磷酸化水平增高。 2)正常状态下,SSeCKS在脑组织中散在分布,高表达于神经元。LPS腹腔注射后,SSeCKS在星形胶质细胞内表达上调。 3)在体外培养的星形胶质细胞细胞中,LPS、IL-1β、TNF-α作用后,细胞内SSeCKS表达水平和磷酸化水平改变;与此同时,SSeCKS在细胞内重新分布:由胞浆向核周迁移,于胞膜足突浓集。PKC的抑制剂可以阻断SSeCKS在细胞内的重排及磷酸化水平的上调。 2. 1)LPS可诱导TNF-α在星形胶质细胞内合成,该过程受到PKC-细胞外信号调节激酶(Extracellular signal regulating kinase,ERK)信号途径的调节。 2)SSeCKS基因沉默可抑制LPS作用后星形胶质细胞内TNF-α的合成和分泌。 3)SSeCKS基因沉默可抑制LPS作用后星形胶质细胞内ERK的活性上调。 3. 1)TNF-α能诱导星形胶质细胞增殖。经典型PKC抑制剂G?6976可大幅度阻断该效应,广谱性的PKC抑制剂G?6983或PI3K抑制剂LY294002几乎能完全抑制星形胶质细胞的增殖效应;而新型PKCδ抑制剂Rotterin无明显抑制作用。 2)星形胶质细胞增殖水平与SSeCKS的相对磷酸化水平高度正相关(r=0.905,P=0.02)。 3)PKC活性的丧失对TNF-α所诱导的Akt的磷酸化无明显影响;同样,抑制PI3K的活性不能影响TNF-α作用后SSeCKS磷酸化水平的上调。PKC-SSeCKS和PI3K-Akt相对独立的影响TNF-α所诱导的增殖信号。 [结论] 1.正常状态下,SSeCKS在脑组织中散在分布。LPS作用后该分子反应性表达水平和活性均发生改变,提示该分子作为一种炎症反应蛋白,参与中枢神经系统炎症反应。 2.炎症因子作用后,SSeCKS在星形胶质细胞内的表达和定位发生改变;其磷酸化水平和细胞内定位的变化依赖于PKC的活性,提示该分子可能参与PKC介导的星形胶质细胞活化信号的传递。 3. LPS可诱导TNF-α在星形胶质细胞内合成和分泌,该过程受到PKC-ERK信号途径的调节。 4. SSeCKS的表达降低可影响LPS作用后星形胶质细胞内ERK磷酸化水平的上调和TNF-α的合成与分泌。提示该分子可能作为一个中介分子影响炎症信号从PKC到ERK的传递。 5. SSeCKS参与调节TNF-α诱导的星形胶质细胞的炎性增殖过程。TNF-α刺激后SSeCKS的广泛磷酸化可能是促成星形胶质细胞的增殖的原因之一。 6. PKC-SSeCKS和PI3K-Akt均能影响TNF-α所诱导的增殖信号的传递,它们之间没有明显的上下游关系,但却能互相牵制,提示PKC-SSeCKS和PI3K-Akt信号通路可能在下游存在交集。
[Abstract]:Background and purpose of research

The inflammatory and pathological processes in the central nervous system ( CNS ) are mainly characterized by the activation of glial cells and infiltration of peripheral inflammatory cells . Astrocytes play an important role in maintaining the homeostasis of the central nervous system .

In recent years , it is suggested that the protein kinase C ( PKC ) signaling pathway plays an important role in the activation of astrocytes and plays an important role in regulating the differentiation , migration of astrocytes and the release of cytokines .

In this study , the expression and localization of SSeCKS after intraperitoneal injection of LPS were analyzed , and the role and mechanism of the molecule in the activation of astrocytes were discussed , and a new idea was provided for the treatment of inflammation of the central nervous system .

Methodology

1 . To establish an inflammatory model of LPS - induced injury in rats .

2 . Using the method of Real - time PCR and Western blot to detect the temporal and temporal changes of SSeCKS expression in brain tissue , the cellular localization of SSeCKS in brain tissue was observed by immunofluorescence double standard , and the correlation between activation of astrocytes and the expression level of SSeCKS was analyzed .

3 . The effects of LPS on SSeCKS expression and subcell localization were detected by means of real - time PCR , Western blot and laser confocal technique , and the correlation between the expression level of SSeCKS and PKC activity was analyzed .

4 . Using molecular biology technique to construct SSeCKS RNA interference plasmid and transfected cells . The effects of interfering SSeCKS expression on the biosynthesis and proliferation of astrocytes in the activation process were analyzed by means of immunofluorescence , Western blot , ELISA , flow cytometry ( FACS ) , immunoprecipitation and so on .

The result is not valid .

1.1 ) In the early stage of LPS - induced injury , SSeCKS was up - regulated in brain tissue , and its phosphorylation level was increased .

2 ) In normal state , SSeCKS was distributed in brain tissue and expressed in neurons . After LPS injection , SSeCKS was up - regulated in astrocytes .

3 ) In cultured astrocytes , the level of SSeCKS expression and the level of phosphorylation were changed after LPS , IL - 1尾 and TNF - 伪 . At the same time , SSeCKS was redistributed in the cells : the cytoplasm was migrated to the nucleus around the nucleus , and the concentration of SSeCKS was concentrated . The inhibitor of PKC could block the up - regulation of the rearrangement and phosphorylation of SSeCKS in the cells .

2.1 ) LPS can induce the synthesis of TNF - 伪 in astrocytes , which is regulated by PKC - extracellular signal regulating kinase ( ERK ) signaling pathway .

2 ) SSeCKS gene silencing can inhibit the synthesis and secretion of TNF - 伪 in astrocytes after LPS .

3 ) The silencing of SSeCKS gene could inhibit the increase of ERK activity in astrocytes after LPS .

3.1 ) TNF - 伪 can induce the proliferation of astrocytes . A typical PKC inhibitor , G ? 6976 , can significantly block the effect , and the broad - spectrum PKC inhibitor G ? 6983 or the inhibitor of kinase ( LY294003 ) can inhibit the proliferation of astrocytes completely ; and the novel PKC 未 inhibitor has no obvious inhibitory effect on Rotterin .

2 ) The proliferation level of astrocytes was positively correlated with the relative phosphorylation level of SSeCKS ( r = 0.905 , P = 0.02 ) .

3 ) The loss of PKC activity had no significant effect on the phosphorylation of TNF - a - induced phosphorylation of SSeCKS . Similarly , the inhibition of PI3 activity did not affect the up - regulation of the phosphorylation level of SSeCKS after TNF - 伪 .

Conclusion

1 . In normal state , SSeCKS is dispersed in brain tissue . After LPS , the expression level and activity of this molecule change , suggesting that the molecule acts as an inflammatory response protein and participates in the inflammatory reaction of the central nervous system .

2 . After the action of inflammatory factors , the expression and localization of SSeCKS in astrocytes changed ; its phosphorylation level and intracellular localization depended on PKC activity , suggesting that the molecule might participate in PKC - mediated activation of astrocytes .

3 . LPS can induce the synthesis and secretion of TNF - 伪 in astrocytes , which is regulated by PKC - ERK signaling pathway .

4 . The reduction of SSeCKS can affect the up - regulation of ERK phosphorylation and the synthesis and secretion of TNF - 伪 in astrocytes after LPS . It is suggested that the molecule may act as an intermediate molecule to influence the transmission of inflammatory signals from PKC to ERK .

5 . SSeCKS is involved in the inflammatory proliferation of astrocytes induced by TNF - 伪 . The extensive phosphorylation of SSeCKS after TNF - 伪 stimulation may be one of the reasons for the proliferation of astrocytes .

6 . PKC - SSeCKS and PI3 - 3 could affect the transmission of the proliferative signals induced by TNF - 伪 . There was no obvious upstream - downstream relationship between them , but they could be drawn to each other , suggesting that the pathway of PKC - SSeCKS and PI3 - 3 signaling might exist in the downstream .

【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【共引文献】