【作者】 赵杰；

【导师】 王明山；

【作者基本信息】 大连医科大学 ， 麻醉学， 2016， 硕士

【摘要】 目的:观察亚低温对脑缺血再灌注青年雄性C56BL6小鼠海马蛋白激酶R样内质网激酶(protein kinase R-like ER kinase,PERK),真核翻译起始因子2α(Eukaryotic translation initiation factor 2α,e If2α)和激活转录因子4(Activing transcription factor 4,ATF4)通路表达,神经行为功能评分以及小鼠海马神经元凋亡的影响,探讨亚低温通过抑制内质网应激减少细胞凋亡,发挥脑保护作用的内在机制,为临床治疗脑缺血疾病提供一定的依据。方法:8-12周龄健康雄性C56BL6小鼠144只,体重20-30g,采用随机数字表法,将其随机分为3组(n=48):假手术组(S组)、脑缺血再灌注手术组(I/R组)、全身亚低温手术组(H组)。采用双侧颈总动脉阻断法(BACCO)建立小鼠前脑缺血再灌注模型,脑缺血15 min后再灌注(I/R组)。S组只将双侧颈总动脉与迷走神经分离但不夹闭作为实验对照组。H组于缺血再灌注即刻向小鼠全身喷洒酒精进行体表降温,术中维持小鼠直肠温32~34℃3 h。分别于再灌注6、12、24、72h时,对所有小鼠进行神经行为学评分,并采用Western blot法测定海马p-PERK、p-e If2a和ATF4的表达;HE染色法计数海马存活神经元数目,TUNEL法测定凋亡神经元数目。结果:与S组相比,I/R组和H组再灌注各时点神经行为学评分明显升高,差距具有统计学意义(P<0.05)。S组海马CA1区神经元形态大致正常,脑组织结构清晰完整,细胞核居中,呈圆形或椭圆形,神经元胞浆丰富,着色为浅紫色。I/R组和H组各灌注时间点海马区存活神经元计数明显减少,细胞外形皱缩,核固缩,核染色质深染增多。大片神经元细胞消失、稀疏、排列紊乱。胞核固缩或溶解碎裂,着色为深紫色。S组几乎看不到凋亡细胞,且各时间点之间差异无统计学意义(P>0.05)。与S组相比,IR组与H组海马CA1区再灌注各时间点,贴壁细胞出现皱缩、变形、脱落;而染色细胞呈现染色质浓缩、边缘化,核膜裂解,染色质分割成块状/凋亡小体。TUNEL染色阳性神经元均明显增加(P<0.05),S组小鼠三个蛋白几乎没有或者少量表达,且在再灌注各时间点S组表达相对值的差异没有统计学意义(P>0.05),I/R组和S组海马区p-PERK、p-e If2α和ATF4蛋白表达量升高,差异具有统计学意义(P<0.05)。与I/R组相比,H组再灌注各时间点神经行为学评分降低,差异具有统计学意义(P<0.05)。第12h、24h和72 h时海马存活神经元计数增加,神经元细胞消失、稀疏、排列紊乱。胞核固缩或溶解碎裂减少,差异具有统计学意义(P<0.05)。6h、12h、24h和72h凋亡神经元计数减少,再灌注各时点p-PERK、p-e If2α和ATF4表达下调,差异具有统计学意义(P<0.05)。结论:1、亚低温可以改善脑缺血再灌注后小鼠神经功能缺损。2、亚低温通过抑制小鼠脑缺血再灌注后内质网应激通路p-PERK、p-e If2a和ATF4的表达,从而减轻脑缺血/再灌注损伤时的内质网应激反应,抑制神经细胞凋亡,从而发挥脑保护作用。更多还原

【Abstract】 Objective: To investigate the effect of mild hypothermia on protein kinase R-like ER kinase(PERK)and Eukaryotic translation initiation factor 2α(e If2α)and Activating transcription factor 4(ATF4)in hippocampus following transient cerebral ischemia/reperfusion(I/R)in young male rats and to evaluate its effects on the neurological behavior scores(NBS)the and apoptosis in hippocampal neurons.Methods: One hundred and forty for young male C56BL6 rats,weighing 20-30 g,were randomly divided into 3groups(n=48 each):sham operation group(group S),I/R group and mild hypothermia group(group H).Cerebral I/R was induced by bilateral common carotid artery occlusion method.and bilateral carotid arteries were occluded for15 min.Sham operation group were exposed bilateral common carotid arteries but not occluded for comparison.The surface cooling was started immediately after reperfusion and maintained for 3 h.During surface cooling,the body temperature was maintained at 32-34℃(rectal).At 6,12,24 and 72 h of reperfusion,neurological behavior scores were evaluated for all the rats.the expression of p-PERK,p-e If2α and ATF4 was determined with Western blot.Survival neurons in CA1 area was counted through HE staining.TUNEL staining was used to count the apoptotic neuronsResults: Compared with the S group,the NES in I / R group and H group were significant increased at each time point of reperfusion,the difference was statistically significant(P<0.05).In S group,the morphology of hippocampal CA1 neurons was roughly normal,the structure of the brain was clear and complete,the nucleus was centered,round or oval,and the cytoplasm was abundant,and the color was light purple.Cell shape shrinkage,nuclear pyknosis and nuclear chromatin stained increased.Large tracts of neurons are lost,sparse,and disorganized.Cell nuclear condensation or dissolution fragmentation,the color is dark purple.There was no significant difference in the apoptotic cells in the S group,and there was no significant difference between the time points(P>0.05).anchorage-dependent cell was shrinking,rounded and shed.The staining cell exhibited chromatin condensation and marginalization,nuclear fragmentation,chromatin is divided into blocks,and apoptotic bodies in the CA1 region of the hippocampus at each time point in group IR and group H compared with group S.the number of TUNEL positive neurons were significantly increased(P < 0.05),and the expression of three proteins of the mice in S groups almost no or a small amount and no significant difference were detected in the proteins expression of reperfusion at each time point among S groups(P > 0.05).The level of protein p-perk,p-e If2 alpha and ATF4 were significant increased at each point in I/R and H group(P<0.05).Compared with the I / R group,the ENS was significantly lower at each point of reperfusion in H group,and the difference was statistically significant(P<0.05).In the first 12 h,24h and 72 h,the survival of the neurons in the hippocampus was increased,and there is a loss in the cells disappeared,sparse and disordered,the nuclear condensation or dissolution were decreased in H group,the difference was statistically significant(P<0.05).The number of apoptotic neurons was significantly lower at 12,24 and 72 h of reperfusion and the expression of ATF4,p-e If2 and p-PERK were down regulated at different time points in group H than in group I/R.the difference was statistically significant(P<0.05)Conclusion: 1.Mild hypothermia could reduce neurological deficits and brain damage after cerebral ischemia/reperfusion in rats,2.Mild hypothermia could inhibit apoptosis signal pathway by down-regulate PERK,e If2α and ATF4 expression in hippocampus following transient cerebral I/R in young male C56BL6 rats,thus reducing endoplasmic reticulum stress reaction,neuronal apoptosis and cerebral I/R injury.更多还原