The Research On Inhibitory Effects Of Silencing α1, 3GT Expression By Lentivirus-mediated RNAi In Mouse Vascular Endothelial Cell

Objective:Investigate the feasibility of inhibitingα1,3GT and Galα(1,3)Gal expression in mouse vascular endothelial cell by lentivirus-mediated RNAi,by usingα1,3GT gene targeting shRNA recombined lentivirus.Methods:The mouse vascular endothelial cell was cultured in vitro by tissue nubbles culyivation.The shRNA specified toα1,3GT mRNA was designed and synthesized in vitro and cloned to the lentivirus vector.EOMA cells were infected by recombined lentivirus.Real-time RT-PCR was used to detect mRNA transcriptional levels ofα1,3GT and immunofluorescence was applied to detect Galα(1,3)Gal antigen level after gene tranfection.Result:The mouse vascular endothelial cell was successfully isolatated and cultured. The shRNA sequences were cloned to the lentivirus vector corrently and the lentivirus was produced successfully.The result of Real-time PCR showed that the mRNA transcription ofα1,3GT was obviously inhibited byα1,3GT-shRNA recombined lentivirus(P＜0.01),while there were no obvious changes and differences among non-shRNA lentivirus group,negative-shRNA control group and control group(P＞0.05).Immunofluorescence showed the same results that Galα(1,3)Gal antigen expression ofα1,3GT-shRNA transfected EOMA was less than that of non-shRNA lentivirus group,negative-shRNA control group and control group(P＜0.01),while there were no obvious changes and differences among the later three groups(P＞0.05).Conclusion:In vitro culture of the mouse vascular endothelial cell by tissue nubbles culyivation is a reliable method,but the passage times are limited.We construct the recombinedα1,3GT-shRNA lentivirus and infect EOMA successfully.It proves thatα1,3GT-shRNA lentivirus could inhibit the expression ofα1,3GT mRNA and Galα(1,3)Gal protein.It indicates that the method using Lentiviral-mediated RNAi to inhibit the expression ofα1,3GT mRNA and Galα(1,3)Gal protein to control HAR is feasible.