亚低温对脑缺血大鼠海马神经元中类泛素蛋白表达的调控  

Mild hypothermia improves the neuroprotection ability of SUMO through binding its target proteins in neurons after cerebral ischemia

作　　者：刘洪良[1] 王彤宇[3] 刘振林[1] 陈镭[1] 杨巍[4] 盛华新 姜忠敏[2] 刘晓智[1] 

机构地区：[1]天津市第五中心医院神经外科,300450 [2]天津市第五中心医院病理科,300450 [3]天津医科大学神经内科 [4]杜克大学多学科神经保护实验室

出　　处：《中华神经外科杂志》2015年第3期294-298,共5页Chinese Journal of Neurosurgery

基　　金：国家自然科学基金（81471175）;滨海新区医药卫生科技项目（2012BWKL001,2013KJXQ03）

摘　　要：目的 研究亚低温对大鼠海马神经元中类泛素蛋白（SUMO）表达的调控作用,探索亚低温对脑缺血后神经元的保护机制.方法 原代培养孕龄18 dSD大鼠的海马神经元,培养后12 d,分为对照组、氧糖剥夺（OGD）组、OGD＋亚低温组.37℃或亚低温（33℃）条件下培养OGD神经元24h.采用Western blot法检测SUMO-1和SUMO-2/3的表达水平,ELISA法检测乳酸脱氢酶（LDH）含量,原位凋亡法检测凋亡细胞数量.取SD大鼠36只,按完全随机化原则分为假手术组、大脑中动脉阻塞（MCAO）组及MCAO＋亚低温组,每组12只.线栓法建立MCAO模型,比较3组模型制作后第1、7、14及21天的神经功能缺损评分;免疫荧光法检测海马区神经元中SUMO-1和SUMO-2/3的表达定位.结果 （1）对照组、OGD组及OGD＋亚低温组的LDH分别为（9.85±2.47） pg/L、（586.29±51.56） μg/L、（317.54 ±42.33） pg/L,细胞凋亡率为（0.27±0.05）％、（29.25 ±6.07）％、（17.44±4.59）％,P值均＜ 0.01.3组间游离及共轭SUMO-1蛋白表达水平差异均无统计学意义;与OGD组比较,OGD＋亚低温组游离SUMO-2/3的表达水平降低（P＜0.05）,共轭SUMO-2/3表达水平升高（P＜0.01）,且均高于对照组（P＜0.01）.（2）第7、14天MCAO＋亚低温组神经功能缺损评分均低于MCAO组（P＜0.05）,至第21天两组差异无统计学意义.MCAO组脑梗死面积为（34.51±7.44） mm2,MCAO＋亚低温组为（23.67-±5.49） mm2 （P ＜0.05）;假手术组、MCAO组及MCAO＋亚低温组SUMO-1的表达相近;与假手术组比较,MCAO组细胞核的SUMO-2/3表达明显增高,MCAO＋亚低温组增高趋势更明显.结论 大鼠亚低温神经保护机制可能与提高脑缺血后SUMO-2/3与靶蛋白的结合能力有关.Objective To study the regulation mechanism of hypothermia on small ubiquitin-related modifier （SUMO）,and explore the protective mechanism of mild hypothermia therapy on cerebral ischemia.Methods The hippocampal neurons were obtained from SD rats with gestational age of 18 days.After cultured 12 days,they were randomly divided into three groups：control group,oxygen glucose deprivation （OGD） group and OGD ＋ mild hypothermia group.These OGD neurons were cultured in 37 ℃ and mild hypothermia （33 ℃） condition for 24 hours respectively.And the expression level of SUMO-1 and SUMO2/3 were detected by western blot method.The lactate dehydrogenase （LDH） content was detected by ELISA,and apoptosis by apoptosis in situ.A total of 36 SD rats were randomly divided into sham operation group,middle cerebral artery occlusion （MCAO） group and MCAO ＋ mild hypothermia group,12 rats in each group.MCAO model was established with thread embolism method,and their neurologic impairment scores were compared among three groups at 1,7,14 and 21day.The expression localization of SUMO-1 and SUMO-2/3 in hippocampus neurons were detected by immunofluorescence assay.Results （1） In control group,OGD group and OGD ＋ mild hypothermia group,the LDL levels were （9.85 ± 2.47） μg/L,（586.29 ± 51.56） μg/L,（317.54 ± 42.33） μg/L respectively,and the apoptosis rates were （0.27 ± 0.05） %,（29.25 ± 6.07） %,（17.44 ± 4.59） % respectively.All the P value was 〈 0.01.The SUMO-1,both free and conjugated protein expression level showed no statistically difference among three groups.Compared with OGD group,the expression level of free SUMO-2/3 protein reduced in OGD ＋ mild hypothermia group,but the conjugated SUMO-2/3 level increased.Both free and conjugated SUMO-2/3 protein level in OGD group and OGD ＋ mild hypothermia group were higher than that in the control group （P 〈 0.01）.（2） The neural function defect score of MCAO ＋ in mild hypothermia group at the 7th and 14th day were lo

关 键 词：脑缺血 低温 人工 泛素类 海马 神经元 大鼠 

分 类 号：R743[医药卫生—神经病学与精神病学]