【作者】 孙景军；

【导师】 叶诸榕； 刘颖；

【作者基本信息】 复旦大学 ， 病理学， 2008， 博士

【摘要】 反应性星形胶质化是星形胶质细胞对各种脑损伤后的修复反应,表现为星形胶质细胞的肥大、增生和GFAP含量增加。星形胶质细胞在中枢神经系统受损后的恢复过程中同时存在有益及有害两方面的作用,一方面星形胶质化可以限制炎症及有害物质扩散,分泌神经营养因子,修复受损组织;同时过度胶质化也会妨碍轴突再生,影响神经功能恢复。因此,实现胶质化的可控性,减少胶质化的负面影响甚有必要。星形胶质细胞分泌的营养因子包括胶质细胞源性神经营养因子（GDNF）,GDNF在中枢神经损害中的治疗作用引人关注。多年来细胞外ATP及其衍生物被认为是一类神经递质。1978年,Burnstock提出嘌呤能受体概念并将其分为P1和P2两类。后来进一步证实并克隆出多种相关受体,其中P2Y₁受体分布广泛,并且发现其在神经系统分布密度较高,P2Y₁受体在神经系统的作用越来越受到重视。已有报道,星形胶质细胞的P2受体可以促进STAT3磷酸化,STAT3活化在星形胶质化中可能起关键作用。P2受体激动剂可以诱导星形胶质化,P2受体拮抗剂可以减轻损伤所致的胶质增生,并发现P2Y(P2Y₁或P2Y₁₂)受体参与了星形胶质细胞的胶质化过程。星形胶质细胞中ERK可以使CREB磷酸化,从而导致其分泌GDNF。GDNF对多种神经元细胞都有营养作用,其在神经损伤后的治疗价值引起了临床医学界的广泛关注。在缺血性脑损伤时,针对P2Y₁受体在星形胶质化及其对GDNF分泌中的作用;这两种过程的相关信号通路以及RAS/ERK途径与这些信号通路之间的关系未见报道。本实验应用大鼠大脑中动脉栓塞后再灌注和原代培养胚鼠脑组织无氧、无营养处理后再恢复正常培养作为短时性缺血的体内、外模型,观察P2Y₁受体在短时性缺血后星形胶质化、及其对GDNF分泌中的作用;进一步探讨了短时性缺血时P2Y₁受体在星形胶质化、GDNF分泌中的相关信号传导途径。研究结果表明阻断P2Y₁受体能够抑制星形胶质细胞合成GFAP,但能促进该细胞分泌GDNF。进一步研究表明星形胶质细胞表面的P2Y₁受体通过JAK/STAT3途径影响GFAP合成,通过AKT/CREB途径影响GDNF分泌。RAS/ERK通路的某些信号分子（如MEK1/2或其下游某些分子ERK1/2等）为这两种途径共同的上游信号分子,说明P2Y₁受体影响合成GFAP和GDFN的分泌具有负相关性,两条信号通路存在串话（crosstalk）。本研究对实现可控性反应性星形胶质化和脑缺血后的功能恢复提供了新思路和手段。第一部分P2Y₁受体对大鼠短时性脑缺血后星形胶质化及其对GDNF分泌的影响目的研究短时性脑缺血后P2Y₁受体对大鼠脑星形胶质化及其对GDNF分泌的影响方法分别以大鼠大脑中动脉栓塞再灌注及对原代培养胚鼠脑组织进行无氧、无营养处理作为短时性脑缺血体内、体外模型;以GFAP表达作为星形胶质化的生物学标志;以MRS2179作为P2Y₁拮抗剂;采用免疫荧光双标记检测P2Y₁及GDNF的分布;采用Western blotting和Real time RT-PCR分别从蛋白和核酸水平检测GFAP的表达变化;采用Elisa检测脑组织总蛋白及培养细胞上清中GDNF的含量。结果在短时性缺血时,阻断P2Y₁受体能抑制星形胶质细胞分泌GFAP（P＜0.05）;阻断P2Y₁受体能促进星形胶质细胞分泌GDNF（P＜0.05）。结论阻断P2Y₁受体能抑制短时性缺血时星形胶质化;阻断P2Y₁受体能促进短时性缺血时星形胶质细胞分泌GDNF。第二部分P2Y₁受体在短时性缺血时星形胶质化及其分泌GDNF的信号传导途径目的研究P2Y₁受体在短时性缺血时星形胶质化及其分泌GDNF的信号传导途径方法采用Western blotting检测P2Y₁受体阻断后,原代培养星形胶质细胞在缺氧、无营养后信号传导途径PI3-K/AKT/CREB及JAK2/STAT3中的活化信号分子的表达变化情况。结果阻断P2Y₁受体,缺氧、无营养后pAKT、pCREB升高,pSTAT3降低;应用AG490抑制STAT3的上游分子JAK2后,pSTAT3及GFAP均降低;应用LY294002抑制PI3-K后,pAKT、pCREB的表达均降低;应用MEK1/2抑制剂U0126后,pAKT、pCREB、pJAK2、pSTAT3的表达均降低。结论短时性缺血时,阻断P2Y₁受体可以阻碍星形胶质化过程,其阻碍胶质化过程通过JAK2/STAT3途径。阻断P2Y₁受体可以促进短时性缺血时星形胶质细胞GDNF分泌;其促进分泌过程可能通过AKT/CREB途径。RAS/ERK途径对这两种信号传导途径都有影响。研究结果表明阻断P2Y₁受体能够抑制星形胶质细胞合成GFAP,但能促进该细胞分泌GDNF。进一步研究表明星形胶质细胞表面的P2Y₁受体通过JAK/STAT3途径影响GFAP合成,通过AKT/CREB途径影响GDNF分泌。RAS/ERK通路的某些信号分子（如MEK1/2或其下游某些分子ERK1/2等）为这两种途径共同的上游信号分子,说明P2Y₁受体影响合成GFAP和GDFN的分泌具有负相关性,两条信号通路存在串话（crosstalk）。本研究对实现可控性反应性星形胶质化和脑缺血后的功能恢复提供了新思路和手段。更多还原

【Abstract】 Reactive astrogliosis is a repair process after various insults to Central Nervous System.Astrogliosis manifests hypertrophy with increase of its GFAP contents as well as hyperplasia of astrocyte.The relevance of astrogliosis remains controversial,especially with respect to the beneficial or detrimental influence of reactive astrocytes on CNS recovery. The reactive astrocytes can secret neurotrophic factors and confine the spread of inflamation and toxic chemicals.At the same time,The formation of a glial scar may interfere with neuronal repair or axonal regeneration in the CNS.Therefore controllable astrogliosis might be an admirable goal:to enhance its beneficial effects of gliosis and to avoid its detrimental effects.Extracellular ATP and its derivants have been considered as transmitters for years. "Purinergic" receptors were first formally recognized and classified as P1 and P2 subtypes by Bumstock in 1978.The widespread and abundant distribution of the P2Y1 receptor within the brain arouses much interests.P2 receptors can activate STAT3 serine 727 phosphorylation in astrocytes and activation of STAT3 maybe play a key role in astrogliosis.Extracellular signal-regulated kinase(ERK) attributes to CREB phosphorylation that leads to GDNF production in cultured astrocytes.The roles of P2Y1 receptor in astrogliosis, STAT3 phosphorylation and GDNF secretion and the related signal pathways under ischemic brain injury remain elucidation.Our study is targeting on the roles of P2Y1 receptor during the process of astrogliosis and GDNF secretion with the temporal right middle cerebral artery occlusion (MCAO) in vivo,and Oxygen-nurturer deprivation of cultured astrocytes in vitro as the model of temporal ischemic injury.The related signal pathways would be investigated.We hope our work will help us to deepen our understanding of related pathophysiological processes and might arouse new therapeutic approach.Part One The effect of P2Y1 receptor on gliosis and GDNF secretion of rat brain astrocytes after temporal ischemiaObjective:The study was aimed to investigate the effects of P2Y1 receptor on the gliosis and GDNF secretion of astrocytes after temporal ischemia in vivo and in vitroMethods:MCAO in vivo and Oxygen-nurturer deprivation in vitro were used as the model of brain temporal ischemic injury.The expression of GFAP as the biomarker of astrogliosis.MRS2179 was used as selective P2Y1 receptor antagonist. The location of P2Y1 receptor and GDNF was observed by double immunofluorescence labeling.Real time RT-PCR and Western blotting were used to measure the mRNA and protein levels of GFAP.Elisa was used to measure the GDNF expression of damaged tissue and the supernate of cultured cells.Results:Inhibition of P2Y1 receptor could reduce the production of GFAP (P＜0.05) and increase the GDNF secretion of astrocytes after the temporal ischemic injury.Summary:Inhibition of P2Y1 receptor could interfere in the astrogliosis and increase the GDNF secretion of astrocytes after the temporal ischemic injury.Part Two The signaling pathways during astrogliosis and GDNF secretion of astrocytes after temporal ischemic injury in vitro Objectives:The purpose is to investigate the signaling pathways during astrogliosis and GDNF secretion of astrocytes after temporal ischemic injuryMethods:Measuring of the level of phosphorylation of signaling molecules involving PI3-K/AKT/CREB and JAK2/STAT3 pathways in the cultured cells after inhibition of the P2Y1 receptor and temporal Oxygen-nurturer deprivation.Summary:Under ischemic injury and blocking the P2Y1 receptor the phosphorylation of AKT and CREB was increased and the phosphorylation of STAT3 was decreased.AG490 could reduce the expression of pSTAT3 and GFAP.LY294002 could reduce the expression of pAKT and pCREB.U0126 could reduce the expression of pAKT,pCREB,pJAK2,and pSTAT3.Conclussions:The study showed that blockage of P2Y1 receptor might decrease the contents of GFAP,while increase the secretion of GDNF of astrocyte.It indicated that synthesis of GFAP and GDNF,influenced by P2Y1 receptor,showed negative correlation.P2Y1 receptor on astrocytic membrane affected GFAP synthesis through JAK2/STAT3 pathway,and influenced GDNF secretion via PI3-K/AKT/CREB pathway respectively.Some molecules,such as MEK1/2 and ERK1/2 in RAS/ERK pathway might be upstream signal molecules of JAK2/STAT3 and PI3-K/AKT/CREB.Crosstalk existed between signal pathways of JAK/STAT3 and PI3-K/AKT/CREB.更多还原