【作者】 赵杰；

【导师】 袁贤瑞；

【作者基本信息】 中南大学 ， 神经外科学， 2009， 博士

【摘要】 脑缺血-再灌注损伤是临床上常见的病理现象,如脑血管病的手术治疗、脑血管意外后的血管自通或者溶栓治疗、脑外伤的治疗、心肺脑复苏治疗等,都可能发生脑缺血-再灌注损伤。寻找有效防治脑缺血-再灌注损伤的方法是当代神经科学的重要课题之一。脑缺血预适应是到目前为止所发现的最强有力的神经内源性保护现象。揭示脑缺血预适应的发生机制并予以利用,对脑缺血-再灌注损伤的防治具有重要意义,也是国内外学者关注的焦点。WDR26基因是最近从大鼠心肌组织中首次克隆的一个在缺血预适应中表达上调的新基因,定位于染色体1q42.12,含14个外显子和13个内含子,ORF为1,545bp,编码514个氨基酸,Northern杂交结果显示其有四个剪切型。采用多组织膜研究发现,WDR26基因在脑组织中高表达。它是一个典型的WD-重复蛋白。WD-重复蛋白家族成员的序列在真核生物不同物种间均高度保守,在信号转导、蛋白运输、染色体修饰、细胞周期调控、细胞凋亡等多个生命过程中具有重要作用。但WDR26的生物学功能目前尚不清楚。为了深入探讨WDR26在脑缺血预适应及脑缺血-再灌注损伤中的生物学功能,本研究首先通过改良四血管阻断法制备大鼠脑缺血预适应及缺血-再灌注损伤模型,使全脑缺血3min,分别再灌注3h、6h、12h、24h,然后再行全脑缺血30min,再灌注24h,深麻醉下快速断头取脑,进行脑水含量测定。结果发现,缺血3min再灌注24h后再行缺血30min再灌注24h,脑水含量较假手术对照组明显减少,而其它实验组的脑水含量与假手术组无明显差异;说明缺血3min在灌注24h对其后的脑缺血-再灌注损伤导致的脑水肿具有明显保护作用。采用real-timePCR检测脑缺血3min再灌注后WDR26基因在脑组织中的表达情况,结果发现,缺血3min再灌注后WDR26基因在脑组织中的表达较假手术对照组明显增高,再灌注6小时达到高峰。上述结果提示WDR26的表达增高可能是脑缺血预适应保护机制的一部分。氧化应激是脑缺血-再灌注损伤的主要损伤机制之一。为了解WDR26对氧化应激所致的神经细胞损伤是否具有保护作用,本研究采用H₂O₂刺激制备氧化应激细胞模型,检测WDR26基因在氧化应激所致SH-SY5Y神经母细胞瘤细胞损伤过程中的表达情况;将该基因的最大开放阅读框架克隆入真核表达载体pCDNA3.1,并转染入SH-SY5Y细胞,建立WDR26过表达细胞株,探讨WDR26过表达对氧化应激所致神经细胞损伤的影响;采用针对WDR26的反义寡核苷酸（antisense phosphorothioate oligodeoxynucleotides,asODNs）转染,探讨WDR26表达抑制对氧化应激所致神经细胞损伤的影响。研究结果发现,WDR26基因在H₂O₂所致氧化应激中表达明显增高;WDR26过表达可明显抑制H₂O₂导致的SH-SY5Y细胞凋亡,提高SH-SY5Y细胞的存活率;相反,采用反义寡核苷酸技术使WDR26的表达降低则能够显著促进H₂O₂导致的SH-SY5Y细胞死亡。上述结果表明,WDR26对H₂O₂所致神经细胞损伤具有明显保护作用。AP-1是由Fos-Jun或Jun-Jun二聚体构成的具有氧化还原敏感性的转录复合体,对于内源性和外源性ROS引发的氧化应激均具有重要调节作用。因此,本研究采用EMSA和荧光素酶报告基因检测了WDR26过表达对H₂O₂所致神经细胞中AP-1活化的影响,结果发现WDR26过表达能显著抑制H₂O₂诱导的AP-1的活化。因此,AP-1活化的抑制是WDR26发挥神经细胞保护功能的可能机制之一。星形胶质细胞是一种最主要的神经胶质细胞,在中枢神经系统中具有重要作用。本研究采用U251-MG星形母细胞瘤细胞探讨了WDR26对氧化应激所致星形胶质细胞损伤的影响。结果发现,H₂O₂能够诱导U251-MG星形胶质母细胞瘤细胞中WDR26表达显著增加;WDR26过表达可明显抑制H₂O₂导致的U251-MG细胞凋亡,提高U251-MG细胞的存活率;相反,采用反义寡核苷酸技术使WDR26的表达降低则能够显著促进H₂O₂导致的U251-MG细胞死亡。这些研究结果强烈提示,WDR26对氧化应激所致星形胶质细胞的损伤具有保护作用。综上所述,本研究首次发现WDR26是一个脑缺血预适应相关基因,它在氧化应激所致神经细胞和星形胶质细胞损伤过程中具有重要保护作用。该发现为阐明脑缺血预适应的细胞内源性保护机制,寻找脑缺血-再灌注损伤的有效防治措施提供了新的线索。更多还原

【Abstract】 Cerebral ischemia-reperfusion injury is a common phenomenon which leads to neuronal death.Cerebral ischemic preconditioning is a powerful protective mechanism.It can prevent neural cells and astrocytes from apoptosis and necrosis induced by cerebral ischemia/reperfusion injury.Therefore,it is important to illustrate the mechanisms of cerebral ischemic preconditioning.WDR26 is identified as a novel gene which is up-regulated during myocardial ischemia/reperfusion.The completed cDNA of WDR26 is 3,729 bp,composed of an ORF of 1,545 bp.The WDR26 protein is 514 amino acids and the relative molecular mass is 58,603 Da（～59 kDa）. Structural analysis reveals that the protein contains a conserved WD-40 region consisting six WD-40 repeats.WDR26 is expressed abundantly in most of tissues,including at a high level in brain.WD-40 repeat proteins play important roles in a variety of cellular functions,including cell growth,proliferation,apoptosis,and intracellular signal transduction. However,little is known about the role（s） of WDR26.In the present study,we firstly used meliorated four-vessel occlusion to copy global cerebral ischemic preconditioning and ischemia-reperfusion model of rat and to observe the protective effects of cerebral ischemic preconditioning on the following cerebral ischemia/reperfusion injury.Compared with the sham control group,the brain water content of experimental group which was treated 24 hours after operation with 30 minutes cerebral ischemia followed by 24 hours reperfusion was reduced.The results indicated the global cerebral ischemic preconditioning and ischemia-reperfusion model of rat was copied successfully.And the expression of WDR26 was significantly up-regualted following 3 minutes cerebral ischemic preconditioning.Secondly,hWDR26 plasmids and asODNs against WDR26 were used to reveal the role of WDR26 in response to oxidative stress in human SH-SY5Y neuroblastoma cells.The results showed that H₂O₂ at 0.5mM induced a marked increase of cell death and significant up-regulation of WDR26 expression.In addition,over-expression of WDR26 significantly supressed H₂O₂-induced SH-SY5Y cell death. asODNs markedly inhibited the de novo biosynthesis of WDR26,which contributed to enhanced cell death induced by H₂O₂.Moreover, over-expression of WDR26 down-regulated the transcriptional activity of AP-1 during H₂O₂-induced SH-SY5Y cell death.These data demonstrated that WDR26 was up-regulated by oxidative stress and played important roles in H₂O₂-induced SH-SY5Y cell death,which might be mediated by down-regulation of AP-1 transcriptional activity.Thirdly,the role of WDR26 in response to oxidative stress in human U251-MG glioma cells was detected.The results showed that H₂O₂ at 0.5mM induced a significant increase of cell death and up-regulation of WDR26 expression.In addition,over-expression of WDR26 significantly supressed H₂O₂-induced cell injury,asODNs against WDR26 enhanced cell death induced by H₂O₂.These data demonstrated that WDR26 was up-regulated by oxidative stress and played important roles in H₂O₂-induced U251-MG cell death.In together,for the first time we determined that WDR26 was a cerebral ischemic preconditioning related gene and it played important roles in protection from the cell death induced by oxidative stress in neuronal cells and astrocytes.The exact mechanisms are still under active investigation.更多还原