【作者】 柯荣湖；

【导师】 叶诸榕； 刘颖；

【作者基本信息】 复旦大学 ， 病理学， 2009， 博士

【摘要】 前言反应性胶质化的主要表现为星形胶质增生和胶质纤维酸性蛋白(GFAP)表达增加。它是中枢神经系统中各种脑损伤后的修复反应,但过度的胶质化反应将形成胶质瘢痕进而影响神经修复和轴突再生。因此,研究胶质化的调控以减少胶质化反应的负面影响显得尤为重要。腺苷A1受体(A1R)和A2a受体(A2aR)是4种腺苷受体(A1、A2a、A2b及A3)中的最主要的2种亚型,它们作用于星形胶质细胞后可产生不同效应:AIR拮抗剂可部分抑制ATP对胶质化反应的诱导作用,提示A1R可能抑制反应性胶质化;而在A2aR的相关研究中发现,A2aR基因敲除小鼠的GFAP表达减少;A2aR拮抗剂Sch58261可部分逆转bFGF对胶质化反应的诱导作用。这些体内外研究提示A2aR可能诱导反应性胶质化,具有与A1R相拮抗的作用。缺血缺氧损伤后,A1R与A2aR是否参与胶质化?其机制如何?此外,体内外研究证实,在中枢神经系统(CNS)中A1R与A2aR可产生相互拮抗的生物学作用,如脑缺血后A1R具有神经保护作用,而A2aR对神经修复具有损伤作用。那么在缺血缺氧后的反应性胶质化中,二者是否可产生相互拮抗的作用?二者是否存在相互作用?均尚未见报道。为了探讨上述问题,本课题通过星形胶质细胞的糖氧剥夺建立体外缺血缺氧模型,采用Western blot、基因转染以及免疫共沉淀等实验方法,观察A1R与A2aR在缺血缺氧/再灌条件下对反应性胶质化的影响,并探讨与之相关的信号通路。此外,本课题还初步探讨了A1R与A2aR之间的相互作用及其相关的信号通路,以期能够为反应性胶质化的调控及其机制提供实验依据。第一部分腺苷A1受体在反应性胶质化中的作用及其机制目的研究体外糖氧剥夺后A1R在反应性胶质化中的作用及其相关的信号通路方法培养大鼠星形胶质细胞系RBA-2细胞,以GFAP表达作为胶质化反应的生物学标志;采用Western blot方法,观察A1R激动剂CPA与拮抗剂DPCPX在糖氧剥夺基础后对GFAP表达影响,并用Western blot方法初步探讨Akt以及Jak2/STAT3信号通路的改变。结果糖氧剥夺后,A1R激动剂CPA能抑制RBA-2细胞中GFAP蛋白表达,A1R拮抗剂DPCPX可逆转CPA对GFAP表达的影响;CPA呈剂量依赖性上调p-Akt水平,而DPCPX预处理可抑制CPA对p-Akt的影响。阻断Akt可上调GFAP表达,并逆转CPA对GFAP蛋白表达的抑制作用。CPA作用于RBA-2细胞后,可使p-Jak2水平下降但对p-STAT3水平无明显影响。阻断Akt可上调p-Jak2水平,并逆转CPA对p-Jak2的抑制作用。小结糖氧剥夺基础后,A1R通过Akt磷酸化抑制胶质化反应,且Akt与Jak2信号通路间存在crosstalk。第二部分腺苷A2a受体在反应性胶质化中的作用及其机制目的研究体外糖氧剥夺后,A2aR在反应性胶质化中的作用及相关的信号通路(Akt/NF-κB)。方法将真核表达质粒pEYFP-A2aR稳定转染至RBA-2细胞中,筛选出A2aR高表达克隆(A2aR~+细胞):采用Western blot和FACS,分别检测A2aR~+细胞和转染空载质粒的RBA-2细胞(A2aR~-细胞)GFAP表达和细胞周期。A2aR的抑制剂Sch58261,Akt抑制剂单独使用或二者合用后,观察A2aR~+细胞的GFAP表达和细胞周期;用Western Blot检测Sch58261对Akt信号通路的影响,并观察阻断Akt后,Sch58261对GFAP表达、细胞周期以及NF-κB信号通路的影响。结果Western blot和FACS结果显示,在常氧和糖氧剥夺条件下A2aR转染均可使GFAP表达上调以及S期细胞比例增加,该作用在糖氧剥夺后更明显。A2aR阻断剂Sch58261可抑制GFAP蛋白表达并降低S期细胞比例,Sch58261还可呈剂量依赖性上调p-Akt水平;阻断Akt可逆转Sch58261对GFAP蛋白表达和细胞周期的影响。Western blot结果还显示,Sch58261下调A2aR~+细胞胞浆中的P65但上调胞核中P65水平,且A2aR~+细胞胞浆中p-IκBα/IκBα比值减小,Akt抑制剂可逆转Sch58261对P65和p-IκBα/IκBα的影响。小结糖氧剥夺后,A2aR通过Akt去磷酸化诱导胶质化反应,该过程与P65蛋白的核转位有关,而后者受p-IκBα/IκBα比值调控。第三部分腺苷A1与A2a受体之间的相互作用及其相关的信号通路目的研究体外糖氧剥夺后,A1R与A2aR之间的相互作用,并对该作用所涉及的信号通路进行初步探讨。方法培养A2aR~+细胞,采用免疫共沉淀的方法,定性检测A1R与A2aR间的相互作用;采用Western blot方法,检测糖氧剥夺后A1R拮抗剂DPCPX对A2aR以及GFAP表达的影响并初步探讨该作用所涉及的信号通路(Jak2、Akt)。结果免疫共沉淀结果显示A1R与A2aR间存在相互作用;Western blot结果显示糖氧剥夺后,A1R拮抗剂DPCPX呈剂量依赖性上调A2aR以及GFAP蛋白表达;DPCPX还可抑制A2aR~+细胞中p-Akt以及p-Jak2水平。小结Akt与Jak2信号分子可能参与调节A1R与A2aR间存在相互作用。结论1.体外糖氧剥夺后,A1R通过Akt磷酸化进而抑制胶质化。抑制Akt可上调p-Jak2。提示Akt可能通过p-Jak2介导A1R抑制胶质化。2.体外糖氧剥夺后,A2aR通过Akt去磷酸化诱导胶质化反应,该过程与P65蛋白的核转位有关,而后者受p-IκBα/IκBα调节,提示A2aR可能通过Akt/NFκB信号通路诱导反应性胶质化。3.A1R与A2aR存在相互作用,阻断A1R可上调A2aR蛋白表达,还可下调p-Akt与p-Jak2水平,提示Akt与Jak2信号分子可能参与调节这两种受体间的相互作用。更多还原

【Abstract】 IntroductionGliosis is characterized with astroglial hyperplasia and increasing GFAP expression.Although gliosis is a repair response to various injuries in central nervous system(CNS),excessive gliosis may interfere with neuronal repair and axonal regeneration by glial scar in the CNS.Thus,further understanding of mechanisms of gliosis will facilitate controllable gliosis to attenuate its negative aspects.Among adenosine receptors(A1,A2a,A2b,and A3),A1R and A2aR are two main receptors.They exhibit different effects on astrocytes:A1R induces apoptosis in astrocytoma and its antagonist partly blockades ATP-induced gliosis,suggesting A1R may prevent gliosis.However,reports on A2aR showed GFAP expression was attenuated in A2aR-knockout mice and A2aR inhibitor Sch58261 partly reversed gliosis by bFGF,indicating A2aR may induce gliosis and has reverse roles from A1R. Whether A1R and A2aR are involved in gliosis after ischemia? What mechanisms induced by those two receptors are involved in gliosis after ischemia? Observations in vivo and vitro showed that there were antagonistic functions between the two receptors in CNS.Take ischemia for example:A1R exhibited neuroprotection while A2aR had deleterious role to neural restore.Do the two receptors have antagonistic roles in gliosis after ischemia? Is there interaction of the two receptors? These questions remain elucidation.In this study,we performed oxygen-glucose deprivation(OGD) to mimic ischemic injury in vitro and used molecular biologic technologies including Western blot and transfection to examine the role of A1R and A2aR in gliosis after ischemia/reperfusion and its related signal pathways.Moreover,interaction of A1R and A2aR was investigated by immunoprecipation and Western blot.The purpose of our research is to provide new experimental data for enriching the knowledge on the regulation of gliosis. PartⅠThe role of A1 receptor in gliosisObjective:To explore the role of A1R in the gliosis induced by OGD in vitro and its relative signal pathway.Methods:GFAP expression was used as the biomarker of gliosis and its expression was examined by Western blot after RBA-2 astrocytes were treated with A1R agonist CPA or antagonist DPCPX after exposed to oxygen-glucose deprivation(OGD). Moreover,signal pathways including Akt and Jak2/STAT3 were detected by Western blot.Results:A1R agonist CPA prevented GFAP protein expression,whereas A1R antagonist DPCPX upregulated GFAP expression and resversed the effect of CPA. CPA increased the level of phospho-Akt in dose-dependent manner.The increase in phospho-Akt was attenuated by pretreatment of DPCPX.Akt inhibitor increased GFAP expression and reversed the effect of CPA on GFAP expression.CPA treatment decreased phospho-Jak2 but has no effect on phospho-STAT3.Akt inhibition increased phospho-Jak2 and reversed the effect of CPA on phospho-Jak2.Summary:A1R inhibites gliosis via Akt phosphorylation after OGD.There is crosstalk between Akt and Jak2 signal pathways.PartⅡThe role of A2a receptor in gliosisObjectives:To investigate the role of A2aR in the gliosis after OGD and its related signal pathway.Methods:Eukaryotic expression plasmid pEYFP-A2aR was stably transfected into RBA-2 astrocytes by lipofectin.Western blot and immunofluorescence analysis were performed to verify the transfection.Western blot and fluorescence-activated cell sorter(FACS) were used to analyse the GFAP expression and cell cycles respectively in cells transfected with pEYFP-A2aR(A2aR~+cells) or pEYFP(A2aR~-cells) after OGD.GFAP expression and cell cycles were examined after A2aR~+ cells were treated with A2aR antagonist Sch58261,Akt inhibitor or in combination.Akt/NF-κB pathway was detected by Western blot after A2aR~+ cells were treated with Sch58261,Akt inhibitor or in combination.Results:Both in normal or OGD condition,A2aR~+ cells increased GFAP expression and the percentage of cells in S-phase.The results were more obvious after cells were exposed to OGD.With the increase in phospho-Akt,A2aR antagonist Sch58261 inhibited GFAP expression and decreased the percentage of cells in S-phase.Akt inhibitor reversed the effect of Sch58261 on GFAP expression and cell cycles. Sch58261 downregulated cytoplasmic P65 in A2aR~+ cells while it upregulated nuclear P65.The ratio of phospho-IκBαand IκBαwas decreased after A2aR~+ cells were treated with Sch58261.Akt inhibitor reversed the effect of Sch58261 mentioned above.Summary:A2aR induces gliosis via Akt dephosphorylation after OGD.The procession is associated with P65 translocation regulated by the ratio of phospho-IκBαand IκBα.PartⅢThe interaction of A1 receptor and A2a receptorObjectives:To investigate the interaction of A1R and A2aR and its related signal pathway after OGD.Methods:Immunoprecipitation was used to analyse the interaction of A1R and A2aR in A2aR~+cells.Protein expressions including A2aR,GFAP,Jak2 and Akt signal molecular were detected by western blot after A2aR~+ cells were treated with A1R antagonist DPCPX following OGD.Results:Immunoprecipitation result showed the interaction between A1R and A2aR. A1R antagonist DPCPX induced the expression of A2aR and GFAP in dose-dependent manner after A2aR~+ cells were exposed to OGD.DPCPX downregulated phospho-Akt and phospho-Jak2 in A2aR~+ cells exposed to OGD.Summary:The interaction between A1R and A2aR may be regulated by Akt and Jak2 pathways after OGD. Conclusions1.A1R inhibites gliosis by Akt phosphorylation that can upregulate phospho-JAk2, suggesting Akt mediates A1R-inhibited gliosis via phospho-JAk2 after cells are exposed to OGD.2.A2aR induces gliosis by Akt dephosphorylation,which is associated with P65 translocation regulated by the ratio of phospho-IκBαand IκBα.These results indicate A2aR may induce glosis by Akt/NFκB pathway.3.There is interaction between A1R and A2aR.A1R inhibition can upregulate the expression of A2aR and GFAP but downregulated both phospho-Akt and phospho-Jak2,suggesting interaction of the two receptors may be regulated by Akt and Jak2 pathways.更多还原