CREB-shRNA对氧糖剥夺/复氧皮层神经元线粒体形态和凋亡的影响  

Effect of CREB-shRNA on mitochondrial morphology and cell apoptosis in OGD/R-induced cortical neurons

作　　者：王来[1] 楚方旋 耿慧霞 董瑞瑞 祝世功[3] WANG Lai;CHU Fang-xuan;GENG Hui-xia;DONG Rui-rui;ZHU Shi-gong(School of Life Science,Henan University, Kaifeng 475001, China;School of Nursing and Health Sciences, Henan University, Kaifeng 475001, China;Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing 100191 , China)

机构地区：[1]河南大学生命科学学院,河南开封475001 [2]河南大学护理与健康学院,河南开封475001 [3]北京大学医学部生理与病理生理学系,北京100191

出　　处：《中国病理生理杂志》2017年第8期1487-1493,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81471254;No.81171081);河南省基础与前沿技术研究计划(No.162300410102);河南省博士后科研资助(No.2015051);河南省高等学校重点科研项目(No.15A180031;No.16A330001)

摘　　要：目的:探讨沉默CREB基因对氧糖剥夺/复氧神经元线粒体形态及细胞凋亡的影响。方法:3条靶向CREB基因的shRNA片段插入慢病毒载体pLenti Lox3.7(PLL),与包装质粒psPAX2和pMD2.G共转染293T细胞包装病毒颗粒后感染原代皮层神经元,Western blot检测CREB蛋白的沉默情况;分别用Mito Tracker red、TUNEL和Western blot实验检测氧糖剥夺/复氧诱导CREB基因沉默后神经元的线粒体形态、细胞凋亡和Bcl-2、Bax的表达情况。结果:pLL-CREB-shRNA1为最有效的shRNA,其抑制皮层神经元CREB基因表达率高达80%。CREB基因沉默促进氧糖剥夺/复氧诱导的皮层神经元线粒体向点状、片段化形态改变,同时降低Bcl-2表达、促进Bax的表达并加重细胞凋亡(P<0.05)。结论:CREB-shRNA重组慢病毒载体可特异性抑制神经元CREB基因的表达,CREB基因沉默加重氧糖剥夺/复氧诱导的皮层神经元线粒体形态改变,促进细胞凋亡。AIM:To construct recombinant lentiviral vector with short hairpin RNA(shRNA)ofCREBgene,and to investigate the effect ofCREBgene silencing on mitochondrial morphology and cell apoptosis in oxygen-glucose deprivation/reoxygenation(OGD/R)-induced cortical neurons.METHODS:Three lentiviral vectors pLentiLox3.7(PLL)inserted shRNA fragments targetingCREBgene were co-transfected with the packaging plasmids psPAX2and pMD2.G to the293T cells,and the virus particles,which was infected with the primary cortical neurons,was encapsulated.The protein expression of CREB was detected by Western blot.The mitochondrial morphology,cell apoptosis and the expression of Bcl-2and Bax were evaluated by the methods of MitoTracker red,TUNEL and Western blot in OGD/R induced cortical neurons afterCREBgene silencing.RESULTS:The pLL-CREB-shRNA1was the most effective shRNA,which inhibited80%CREBgene expression in the cortical neurons.The mitochondrial was appeared dot and fragment morphology in OGD/R induced cortical neurons with transfected pLL-CREB-shRNA1plasmid.In addition,the expression of Bcl-2was decreased,the expression of Bax,and the apoptosis of the neurons were increased by tranfected with pLL-CREB-shRNA1.CONCLUSION:CREB shRNA recombinant lentiviral vector specifically inhibits the expression ofCREBgene.CREBgene silencing promotes the cell apoptosis and mitochondrial morphological changes in the cortical neurons induced by OGD/R.

关 键 词：CREB基因 重组慢病毒载体 神经元 氧糖剥夺/复氧 线粒体 

分 类 号：R363.21[医药卫生—病理学]