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

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(P0.05)。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。

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

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 psPAX2 and pMD2.G to the 293T 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-2 and 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-shRNA1 was the most effective shRNA, which inhibited 80% 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-shRNA1 plasmid. In addition, the expression of Bcl-2 was 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.