发育期大鼠反复惊厥持续状态后分子伴侣介导的自噬表达的变化  

Changes in molecular chaperone-mediated autophagy expression of developmental rats after recurrent-status seizures

作　　者：崔盈[1] 倪宏[2] 王春红[1] 徐华 刘月影[1] Cui Ying;Ni Hong;Wang Chunhong;Xu Hua;Liu Yueying(Department of Pediatrics,Affiliated Hospital of Jiangnan University,Wuxi 214062,Jiangsu Province,China;Department of Neurology,Children′s Hospital of Soochow University,Suzhou 215000,Jiangsu Province,China)

机构地区：[1]江南大学附属医院儿科,江苏无锡214062 [2]苏州大学附属儿童医院神经内科,江苏苏州215000

出　　处：《中华实用儿科临床杂志》2021年第14期1102-1107,共6页Chinese Journal of Applied Clinical Pediatrics

基　　金：无锡市医学重点人才项目(ZDRC017);无锡市"双百"中青年医疗卫生拔尖人才项目(BJ2020042)。

摘　　要：目的观察反复惊厥持续状态后海马神经元中分子伴侣介导的自噬分子的表达及其与惊厥后脑损伤的关系。方法取日龄7 d的SD大鼠,采用简单随机分组方法分为对照组(6只)及惊厥组(39只),惊厥组反复吸入三氟乙醚诱导大鼠的惊厥持续状态,1次/d,30 min/次,连续诱导7 d。成功建立惊厥模型共30只(弃去建模失败9只),采用简单随机分组方法分为末次惊厥后0 h、1.5 h、3 h、12 h、24 h组,每组6只。采用Western blot法及反转录聚合酶链反应(RT-PCR)法观察海马神经元中分子伴侣介导的自噬标记分子[热应激同源蛋白70(Hsc70)、溶酶体相关膜蛋白2a(LAMP-2a)、热休克蛋白(HSP)40及HSP90]的表达,原位末端标记法(TUNEL)检测细胞凋亡。结果1.RT-PCR及Western blot显示:与对照组相比,分子伴侣Hsc70表达量在末次惊厥后1.5 h开始升高,持续至惊厥后24 h(P<0.05);HSP90在末次惊厥后立即升高,持续至惊厥后24 h(P<0.01);HSP40及LAMP-2a表达量在末次惊厥发作后也呈现高表达(P<0.05)。2.TUNEL法显示:与对照组(15.16±2.48)/40倍视野相比,惊厥组在末次惊厥后3 h(36.33±5.16)/40倍视野、12 h(44.83±4.83)/40倍视野、24 h(54.83±7.16)/40倍视野,大鼠海马CA1区凋亡细胞数明显增多(均P<0.01)。3.Pearson相关性分析显示发育期反复惊厥持续状态后大鼠海马CA1区的细胞凋亡与分子伴侣标记分子表达呈正相关(Hsc70:r=0.734,P=0.001;LAMP-2a:r=0.790,P<0.001)。结论发育期大鼠反复惊厥持续状态发作后,存在多个分子伴侣介导的自噬表达升高,并与细胞凋亡呈正相关,可能参与了发育期反复惊厥发作后脑损伤过程。Objective To observe the expression of molecular chaperone-mediated autophagy in hippocampal neurons and its relationship with brain injury after recurrent-status seizures.Methods Seven-day-old SD rats were divided into two groups according to simple randomization:the control group(NS group,6 rats)and the recurrent-seizure group(RS group,39 rats).Rats in the RS group were subjected to recurrent seizures after repeated inhalation of flurothyl,with 30 minutes once each day for consecutive 7 days.A total of 30 convulsive models were successfully established(9 rats that failed to establish models were discarded),and they were further divided into 0 h,1.5 h,3 h,12 h and 24 h after the last seizure according to simple randomization,with 6 rats in each group.Western blot and reverse transcription-polymerase chain reaction(RT-PCR)were adopted for the observation of the expression of molecular chaperone-mediated autophagy markers[heat shock cognate protein 70(Hsc70),lysosome-associated membrane protein type 2a(LAMP-2a),heat shock protein 40(HSP40)and heat shock protein 90(HSP90)]in hip-pocampal neurons,and apoptosis was detected by TdT-mediated dUTP nick-end labeling(TUNEL).Results(1)RT-PCR and Western blot showed that,compared with the NS group,the expression of Hsc70,as a molecular chape-rone,started to increase at 1.5 h and continued until 24 h after the last seizure in the RS group(P<0.05).HSP90 increased immediately after the last seizure and lasted until 24 h after the seizure(P<0.01);the expression of HSP40 and LAMP-2a also showed high expression after the last seizure episode(P<0.05).(2)The TUNEL method showed that the number of apoptotic cells in the hippocampal CA1 region increased significantly at 3 h(36.33±5.16)/40 field,12 h(44.83±4.83)/40 field and 24 h(54.83±7.16)/40 field after the last seizure compared with NS group(15.16±2.48)/40 field(P<0.01).(3)Pearson correlation analysis showed that the level of apoptosis in hippocampal CA1 region of rats after recurrent seizures was positively correlated with th

关 键 词：发育期大鼠 反复惊厥持续状态 分子伴侣介导的自噬 细胞凋亡 

分 类 号：R742.1[医药卫生—神经病学与精神病学]