作 者：
宋志琦;朱婷;王进;杨威;崔永勇;周向梅;杨利峰;赵德明
单 位：
福建农林大学动物科学学院;军事医学科学院实验动物中心;中国农业大学动物医学院国家动物海绵状脑病实验室
关键词：
神经元限制性沉默因子;朊病毒病;PrP106-126;原代神经元;神经元死亡;神经保护作用
摘 要：
为检测神经元限制性沉默因子(REST)蛋白过表达或干扰对由PrP106-126毒性多肽引起的原代神经元死亡的影响,首先通过脂质体转染法将已经构建好的pCMV-HA-REST质粒转染原代神经元,或利用靶向REST的小干扰siRNA技术干扰原代神经元中REST蛋白的表达。用PrP106-126毒性多肽刺激构建好的REST过表达或干扰的原代神经元,利用Annexin V-FITC试剂盒检测细胞活性;利用TUNEL和Hoechest试剂盒,在激光共聚焦显微镜下直接观察细胞凋亡情况;在透射电镜下观察原代神经元亚细胞结构的变化和损伤情况;使用JC-1线粒体膜电位试剂盒检测线粒体膜电位的改变,同时检测凋亡相关蛋白FOXO1、细胞色素C以及Caspase-3的变化情况。结果显示,受到多肽刺激后,REST的过表达可减轻由毒性多肽引起的神经元死亡、神经元空泡化、细胞器损伤,抑制线粒体膜电位的改变,维持促存活蛋白FOXO1的表达,阻止线粒体向胞浆中释放细胞色素C以及Caspase-3的激活。相应地,干扰REST后,可加剧毒性多肽对神经元的损伤并抑制FOXO1的表达。这表明REST蛋白的过表达可缓解由PrP106-126毒性多肽引起的神经元死亡,对神经元起保护作用,为进一步阐释REST对朊病毒及相关神经退行性疾病引起的病理性损伤的治疗作用和神经保护机制提供依据。
译 名：
REST alleviates neurotoxic prion peptide PrP106-126-induced primary cortical neuronal death
作 者：
SONG Zhi-qi;ZHU Ting;WANG Jin;YANG Wei;CUI Yong-yong;ZHOU Xiang-mei;YANG Li-feng;ZHAO De-ming;National Animal Transmissible Spongiform Encephalopathy Laboratory,College of Veterinary Medicine,China Agricultural University;College of Animal Science,Fujian Agriculture and Forestry University;Laboratory Animal Center,Academy of Military Medical Science;
关键词：
RE1-silencing transcription factor;;prion diseases;;PrP106-126;;primary cortical neurons;;neuronal death;;neuroprotection
摘 要：
To investigate effect of overexpression or knockdown of RE1-silencing transcription factor(REST)on PrP106-126-induced primary cortical neurons(PCCN)death.TUNEL assay,Hoechst staining and Annexin V-FITC/PI apoptosis assay were used to examine the cell viability after the overexpression or knockdown of REST in PrP106-126-treated PCCN.The morphological changes of PCCN exposed to PrP106-126 were observed by transmission electron microscopy(TEM).The JC-1 mitochondrial transmembrane potential(MTP)assay,immunofluorescence and Western blot analysis were used to assess the potential downstream mechanism of REST acting as a neuroprotective factor.The results showed that overexpression of REST reduces pathological damage and abnormal biochemical alterations of neurons induced by PrP106-126 and maintains neuronal viability by stabilizing the level of pro-survival protein FOXO1 and inhibiting the permeability of the mitochondrial outer membrane,release of cytochrome C from mitochondria to cytoplasm and the activation of Capase-3.Conversely,knockdown of REST exacerbates morphological damage and inhibits the expression of FOXO1.REST acts as a neuroprotective regulator in PrP106-126-induced neuronal death by repressing pro-apoptotic proteins and/or promoting the expression of anti-apoptotic proteins,which may be important for the basic regulatory mechanism of neuron survival in prion diseases and associated neurodegenerative diseases in vivo.
