Lentiviral-mediated ShRNA Silencing Of Notch-1 Inhibits Expression Of Notch Signaling Pathway In Rat Bone Marrow Mesenchymal Stem Cells

????:Lentiviral-mediated shRNA silencing of Notch-1inhibits expression of Notch signaling pathway in rat bone marrow mesenchymal stem cellsObjective:To investigate the role of Notch signaling pathway in bone marrow mesenchymal stem cells in the construction of engineered myocardial-like tissue vascular network.Methods:The lentivirus expression vector was constructed by pLV[shRNA]-EGFP:T2A:Puro-U6]{rNotch1[shRNA]₁9 nt}?PLV-Notch?and pLV[shRNA]-EGFP:T2A:Puro-U6>Scramble_shRNA?PLV-Scramble?and packaged with the lentivirus vector.The mixture was co-transfected into HEK-293 T cells.The lentivirus suspension was collected and the titer of recombinant virus was detected by quantitative PCR.The constructed PLV-Notch and PLV-Scramble were infected with bone marrow mesenchymal stem cells respectively.The expression of enhanced green fluorescent protein gene and target gene rNotch1 in the transfected cells was identified by fluorescence microscopy,RT-qPCR and western blot.Results:The titer of lentivirus vector was more than 1×10⁸ TU/mL.The mature cell lines were screened.The fluorescence rate of the two transfection groups was 80%,and the expression of rNotch1 gene in the interference group was 75.2%of that in the scrambled group.Conclusion:Lentiviral-mediated rNotch1 gene transfection of bone marrow mesenchymal stem cells was successfully constructed and could silence the expression of Notch signal pathway.????:Co-culture of engineered BMSCs with rat EPCs supernatant and the mechanism of interaction between Notch signaling pathway and vascular endothelial growth factor signaling pathway Objective: In this study,we hope to provide theoretical support and data basis for the construction of suitable engineered myocardial tissue through the study of engineered vascular tissue in vitro and the mechanism of Notch and vascular endothelial growth factor related signaling pathway.Methods: The experiment was divided into three groups: stable transfection?interference group?which was constructed in the first part,negative transfection?NC group?and BMSCs?blank group?.WB and RT-PCR were used to detect their protein and gene expression in Notch signaling pathway to determine the effect of down-regulation of Notch gene expression on the construction of experimental engineered tissue cells.The interaction between down-regulation of Notch gene expression and endothelial progenitor cells?EPCs?supernatant was determined by co-culture and proliferation experiments.The stable transfection was detected separately,and the expression of NICD,VEGFA,NICD and Hes genes were detected during co-culture with EPCs supernatant.The results were obtained by Gel-Pro Analyzer software and statistics.Results: The expression of NICD in the stable screening strain of Notch was 0 after the interference was down-regulated,and the effect of Lentivirus on Notch gene was obvious.The results of electrophoresis showed that the size of PCR products was correct and there was no heterozygosity.Compared with NC group,NICD gene expression was significantly inhibited in stable transfection interference group.The expression of vascular endothelial growth factor?VEGF?in interference group was higher than that in blank group and NC group.Conclusion: 1.Lentiviruses significantly interfered with Notch gene,and the expression of Notch gene and subordinate protein NICD was significantly reduced.2..Notch signaling pathway has negative feedback effect on the expression of vascular endothelial growth factor pathway,down-regulates the expression of vascular endothelial growth factor pathway,increases the expression of vascular endothelial growth factor pathway,and actively proliferates cells.3.Notch and VEGF signaling pathways interact in the regulation of blood vessels in engineered tissues.Targeting these signaling pathways may provide new ideas and methods for the further development of engineered tissues with clinical therapeutic effects.