The Interference Effect Of TGFβ 1 Small Interference RNA On Lung Fibroblast Exposed To Hyperoxia In Fetal Rats

OBJECTIVE:To observe the interference effect of small interference RNA on lung fibroblast cultured in vitro exposed to hyperoxia in fetal rats,we study the regulation effect of TGFβ1siRNA on TGFβ1 gene expression to establish the experimental basis of RNA interference in vivo and to provide the new methods to prevention and cure chronic lung disease of premature infants.METHODS:We primary culture lung fibroblast in 19 days' gestation age Sprague-Dawley fetal rats,depurate and identity the cells,then establish the model of hyperoxia induced lung fibroblast injury.Three 21-nucleotides siRNA targeting the TGFβ1 mRNA sequence and constructing the green fluorescence plasmid DNA of expressingTGFβ1 siRNA were transfected into those cells model above.To determine the effect of these siRNAs,the positive plasmid vectors of TGFβ1 were transfected into these cells with the JetFEI,besides using negative and null vectors were treated as above as controls.After 24,48 and 72 hours,the preliminary effect was evaluated with green fluorescence microscope,and then fluorescent quantitation PCR was used to detect the expression of TGFβ1 gene at mRNA level.Moreover,calculating the efficiency of RNA interference.RESULTS:(1)We successfully culture lung fibroblast in 19 days' gestation age Sprague-Dawley fetal rats,depurate and identity the cells,then establish the model of hyperoxia induced lung fibroblast injury.Every 4 19-day-aged fetal rats could averagely get (1.50±0.10)×10~8 lung fibroblast.The purity and vigor of these cells could reach(96±1)% and(97±2)%respectively.(2)These plasmid DNAs were transfected into lung fibroblast by JetPEI for packing,and observing by the green fluorescence microscope.Fluorescence intensity decreased in the TGFβ1 siRNA plasmid groups compared with the negative plasmid groups at 24,48 and 72 hours after transfection.Fluorescence was not observed in the null plasmid groups.(3) Detecting the expression of TGFβ1 gene at mRNA level with fluorescent quantitation PCR.Among every groups,the strongest TGFβ1 gene expression in the group of negative plasmid at 48 hours after transfection(P＜0.05),and the lowest expression of it in the group of TGFβ1 siRNA plasmid at 24 hours after transfection(P＜0.05);the TGFβ1 gene expression was highly knocking down in the groups of TGFβ1 siRNA plasmid compared with groups of negative plasmid at 24,48and 72 hours after transfection(P＜0.01),and their efficiency of RNA interference reduced by degree following times,were97.3%,96.9%and 71.7%respectively.CONCLUSIONS:Transfection with TGFβ1-specific siRNA plasmids could efficiently resulted in sequence-specific decrease in TGFβ1 mRNA levels in hyperoxia induced lung fibroblast injury in fetal rats,and the efficiency of RNA interference reduced by degree following times.