【作者】 汤諹；

【导师】 李树清；

【作者基本信息】 昆明医学院 ， 外科学， 2008， 博士

【摘要】 【目的】观察树鼩血栓性脑缺血及后适应引起的海马神经元凋亡及超微结构改变及其保护效应,研究缺血后适应对脑缺血保护效应的分子机制,探讨PI-3K/Akt信号转导途径在树鼩脑缺血以及缺血后适应所起的生物学作用。【方法】采用光化学反应诱导树鼩血栓性脑缺血,建立脑缺血动物模型,在脑缺血模型成功后立即采用5min夹闭缺血侧颈总动脉5min,再灌注5min,交替进行3个循环以建立缺血后适应模型,应用TUNEL染色法标记脑缺血及缺血后适应的海马CAl区锥体神经元凋亡改变;TTC(2,3,5-triphenyltertrazolium chloride)染色显示动物模型脑梗死范围,电子显微镜下观察不同处理组超微结构改变;应用酶联免疫吸附试验(Enzyme Linked-Immuno-Sorbent Assay,ELISA)定量检测不同处理组在不同时间点磷脂酰肌醇-3激酶(phosphatidylinositol 3-kinase,PI-3K)的重要下游激酶Akt在Akt-Ser-473和Akt-Thr-308位点的活化水平;用免疫组织化学观察Akt[pS473]和Akt[pT308]活化状态及在海马CA1区锥体神经元的定位分布。【结果】脑缺血后,缺血组与缺血后适应组的TUNEL阳性细胞数在缺血侧显著多于对照组(P＜0.01),而在缺血对侧所有实验组TUNEL阳性细胞数与对照组相比均无显著性差异(P＞0.05);在缺血4h时,缺血后适应组TUNEL阳性细胞数较缺血组减少,但无统计学显著差异(P＞0.05),而在缺血24h、72h,缺血后适应组TUNEL阳性细胞数较缺血组显著减少(P＜0.01)。TTC染色显示:缺血后适应能减小梗死面积。电镜观察显示:缺血后适应期间,海马CA1区神经元线粒体和内质网的病理改变明显减轻,细胞水肿改善。ELISA结果显示:脑缺血4h Akt[pT308]水平较对照组显著增高(P＜0.05),但至24h时,Akt[pT308]水平与4h时相比较则显著降低(P＜0.05),而缺血后适应组24h、72h Akt[pT308]水平明显高于对照组(P＜0.05),且72h时,缺血侧后适应组Akt[pT308]活化水平显著高于脑缺血72h组。在不同实验组以及各实验时间点Akt[pS473]水平与对照组相比无显著性差异(P＞0.05)。免疫组化结果显示:海马CA1区锥体神经元可见Akt[pS473]与Akt[pT308]的胞浆、胞膜型表达。【结论】①缺血后适应可减少海马CA1区神经元的凋亡而发挥其神经保护效应。②Akt-Ser-473在脑缺血以及缺血后适应过程中均可活化,而Akt-Thr-308在脑缺血组呈现为一过性活化;缺血后适应组Akt-Thr-308的活化呈现持续、逐渐增强状态,国内外未见报道。由于Akt的活化需Akt-Ser-473与Akt-Thr-308位点的双磷酸化,Akt-Thr-308活化与否决定了PI-3K/Akt途径的激活。③PI-3K/Akt信号转导途径的活化在缺血后适应保护海马CA1区神经元的机制中可能起着重要作用。Akt-Thr-308可能具有作为脑缺血后神经保护药物的作用靶点的潜在价值。更多还原

【Abstract】 OBJECTIVE:To observe the ultrastructural changes of selectively vulnerable hippocampal CA1 neurons to investigate the influence and neuroprotective effect of ischemic postconditioning after ischemic injury,explore molecular mechanisms of protective effect of ischemic postconditioning after cerebal ischemia,and,to inquire what biological role of PI-3K/Akt signal transduction pathway play after ischemic postconditioning and cerebral ischemia in tree shrews.METHODS:Cerebral ischemia was induced by photochemical reaction in tree shrew,and then alternated employed occlusion right common carotid artery 5 min and reperfused 5min at once,carried out 3 circles to establish experimental model of ischemic postconditioning.Using terminal deoxynucleotidyl transferase-mediated uridine 5’-triphosphate-biotin nick end labeling staining(TUNEL)to label apoptosis of pyramid neuron in hippocampus CA1 subfied after cerebral ischemia and ischemic postconditioning,the animal’s brian stained by 2,3,5-triphenyltertrazolium chloride (TTC)to show the infarct size of brain.The ultrastractural impairment changes in different groups were observed under the electronmicroscope.The level of Akt-Ser-473 and Akt-Thr-308 phosphorylation which are the downstream kinase of phosphatidylinositol 3-kinase(PI-3K)were detected quantitatively by Enzyme Linked-Immuno-Sorbent Assay(ELISA)during diferrent time of cerebral ischemia and ischemic postconditioning.The activated expression and distribution of Akt[pS473]and Akt[pT308]in pyramid neuron in hippocampus CA1 in different groups and different phase were surveyed by immunohistochemistry RESULTS:The counts of TUNEL positive neuron of ipsilateral in hippocampus CA1 subfied in cerebral ischemia group and postconditioning group were more than control group significantly(P＜0.01),but the counts of TUNEL positive neuron have no significant difference among all of contralateral groups(P＞0.05).After cerebral ischemia 4 hours,the counts of TUNEL positive neuron in postconditioning group and cerebral ischemia group still.have no statistical difference(P＞0.05),but the counts of TUNEL positive neuron have shown slightly decreased in postconditioning group.After cerebral ischemia 24 hours and 72 hours,the counts of TUNEL positive neuron were significant decreased in ischemic postconditioning groups than ischemic groups(P＜0.01).TTC stained shown that the infarct size of brain was diminished with ischemic postconditioning treatment.The observation with electronmicroscope displayed that the amendment of structure of mitochondria and endoplasmic reticulum in pathological condition of neuron in hippocampus CA1 subfied after ischemic postconditioning.After cerebral ischemia 4 hours,the level of phosphorylation of the Akt protein at threonine residue 308 was significant increased than control group(P＜0.05),however,in the group of after cerebral ischemia 24 hours,the level of phosphorylation of the Akt protein at threonine residue 308 was significant decreased than the group of after cerebral ischemia 4 hours(P＜0.05).In the groups of after ischemic postconditioning 24 hours and 72 hours,the level of phosphorylation of the Akt protein at threonine residue 308 were significant increased than control group(P＜0.05),and the level of phosphorylation of the Akt protein at threonine residue 308 was significant increased in the group of after ischemic postconditioning 72 hours than the group of after cerebral ischemia 72 hours.The level of phosphorylation of the Akt protein at serine residue 473 had not significant difference among all groups(P＞0.05).The result of immunohistochemistry shown that the activated expression of Akt[pS473]and Akt[pT308]were clear in endochylema and membrane in pyramid neuron on hippocampus CA1 subfield.CONCLUSION:①Ichemic postconditioning generate neuroprotective effect after cerebral ischemia by decreased neuron apoptosis in hippocampus CA1 subfied and disminished the infarct size of brain.②Akt protein would be phosphorylated at serine residue 473 ever after cerebal ischemia or ischemic postconditioning,the Akt protein at threonine residue 308 only took place transient phosphorylation during cerebral ischemia but showed activate persistently and increasingly in the group of after ischemic postconditioning.So far,this is first report about the finding.Since the complete activation of Akt needs both serine residue 473 and threonine residue 308 are phosphorylation,so the phosphorylation threonine residue 308 may determine the Akt avtiation.③The activation of PI-3K/Akt pathway may play an important role in neuroprotective effect after cerebral ischemia postcondining.The threonine residue 308 of Akt protein as an important target point of neuroprotective drug after cerebral ischemia may has potential values,deserved to be further explored.更多还原