RÉSUMÉ
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.
RÉSUMÉ
Objective To construct a recombinant lentiviral vector for HCV core-Ant and then study its effect on the transdifferentiation of hepatic stem cells. Methods The HCV core-Ant was obtained by PCR of two primers template with each other and T-vector cloning method,and then subcloned to pLenti6/V5-D-TOPO. The restriction endonuclease and T4 DNA ligase were used to construct the vector. pLenti6/V5-D-HCV core-Ant and the ViraPowerTM PackagingMix (containing three packaging plasmids pLP1,pLP2 and pLP/VSVG) were cotransfected into 293ET cells to produce replication in competent lentivirus after transfection. The viral supernatant on 293T cells was collected. The expression of the lentiviral vector containing HCV core-Ant in Hela cells was measured by immunohistochemistry. Then we constructed the lentiviral vector containing green flurosecent protein by the similar method,and the titers were determined. Results HCV core-Ant was identified and analyzed by restriction enzyme digestion and sequencing,respectively. The expression of the recombinant lentiviral vector plasmid containing HCV core-Ant in Hela cells was confirmed by immunohistochemistry. The 3T3 cells transfected the lentiviral vector containing green flurosecent protein were found to show strong expression of GFP,which confirmed that the four-plasmid system of the lentiviral vector was successfully constructed. Conclusion The recombinant lentiviral vector expressing HCV core-Ant was successfully constructed by molecular cloning and recombination techniques in vitro,which will be beneficial to guiding further study on gene therapy of cancer.