The Interference Effect Of TGFβ1 Small Interference RNA On The Expression Of TGFβ1 In 293 Cells

OBJECTIVE: The effects of RNA interference on TGFβ1 gene were investigated inorder to develop a new tool for studying the roles of TGFβ1 in the development of lungdamage of hyperoxia and to provide the foundation of a potential siRNA therapy of CLDin prematures.METHODS: three 21-nucleotides siRNA targeting the TGFβ1 mRNA sequence weredesigned by myself. Then to construct the green fluorescence plasmid DNA of expressingTGFβ1 siRNA and TGFβ1 fusion genes and identified it. To determine the effect ofthese siRNAs, the positive plasmid vectors of TGFβ1 were transfected into 293 cells withthe aid of lipofectamin, besides using negative and null vectors were treated as above ascontrols. After 24,48 and 72 hours, the preliminary effect was evaluated with greenfluorescence microscope, and then fluorescent quantitation PCR was used to detect theexpression of TGFβ1 gene at mRNA level. Moreover, calculating the efficiency of RNAinterference.RESULTS: (1)The green fluorescence plasmid DNA of expressing TGFβ1 siRNA andTGFβ1 fusion genes were designed and constructed successfully. (2)These plasmid DNAswere transfected int 293 cells by lipofectamin for packing, and observing by the greenfluorescence microscope. Fluorescence intensity decreased in the TGFβ1 siRNAplasmid groups compared with the negative plasmid groups at 24,48 and 72 hours aftertransfection. Fluorescence was not observed in the null plasmid groups. (3) Detecting theexpression of TGFβ1 gene at mRNA level with fluorescent quantitation PCR. Amongevery groups, the strongest TGFβ1 gene expression in the group of negative plasmid at 48hours after transfection (P＜0.05), and the lowest expression of it in the group of TGFβ1siRNA plasmid at 24 hours after transfection(P＜0.05), without TGFβ1 gene expressionin the groups of null plasmid; the TGFβ1 gene expression was highly knocking down inthe groups of TGFβ1 siRNA plasmid compared with groups of negative plasmid at 24,48 and 72 hours after transfection(P＜0.01), and their efficiency of RNA interference reducedby degree following times, were 97.2%,97.1% and 67.7% respectively.CONCLUSION: The results suggested that the TGFβ1 siRNAs designed by my own caninterfere the expreesion of TGFβ1 gene efficiently at 24,48 and 72 hours aftertransfection, and the efficiency of RNA interference reduced by degree following times.