The Studies On Interference Effect Of Short Hairpin RNA On Expression Of TGF-β1 Gene In Human Lung Fibroblast

Objective First,to investigate the effect of transforming growth factor beta 1(TGF-β1)gene expression inhibition by three short hairpin RNA(shRNA)eukaryotic expression plasmids targeting human TGF-β1 gene(shRNA1,shRNA2,shRNA3)in human lung fibroblast(HLF), in order to screen the shRNA plasmid having the most significant interference effect among them.Second,to observe influence of TGF-β1 gene expression inhibited by shRNA on growth of HLF,in order to evaluate the safety of RNAi technology in transfecting shRNA plasmid into HLF for the study about RNA interference(RNAi)apply in gene therapy for pulmonary fibrosis.Methods Three shRNA eukaryotic expression plasmids targeted against human TGF-β1 (shRNA1,shRN2,shRNA3)and corresponding negative control plasmids(Control,Control2,Control3)were analyzed by digestion of restriction endonuclease and DNA sequencing.Then transferred them into HLF by Lipofectamine 2000 respectively which were designated shRNA1 group,shRNAi2 group,shRNA3 group,Control1 group,Control2 group,Control3 group,and set up blank group.After 48 hours,the TGF-β1 mRNA expression in HLF was detected by Real-time Fluorescence Quantitative PCR and the TGF-β1 level in the culture supematant was quantitatively detenrtined by ELISA.To observe morphologic change by light microscope and count the death rate of HLF measured by trypan blue dye.Results After 48 hours transfectd,the TGF-β1mRNA in the shRNA1 group,shRNAi2 group,shRNA3 group were down regulated,and the relative quantity of TGF-β1mRNA was 0.088±0.008,0.022±0.004,0.197±0.030 respectively,compared with blank group,compared with the blank group(P＜0.05).The quantity of TGF-β1 mRNA of HLF about Control groups were not significant difference compared blank group(P＞0.05),and there were not signigicant among them(P＞0.05).The TGF-β1 protein in the in the shRNA1 group,shRNA2 group,shRNA3 group were down regulated respectively,the protein inhibitory rates were 47.7±2.4%,77.4±2.2%,27.3±2.5%respectively(P＜0.001).The quantity of TGF-β1 protein of HLF about Control groups were not significant difference compared blank group(P＞0.05),and there were not signigicant among them(P＞0.05).There were not significant difference between blank group and each transfected group in morphology of HFL(P＞0.05).The death rates of HLF among all shRNA groups measured by trypan blue dye were within the range of 9%～16%,and there were not significant difference between blank group and each shRNA group(P＞0.05).The death rate of HLF about shRNA2 group was not significant difference compared other shRNA group.The death rates of HLF about Control groups were not significant difference compared blank group(P＞0.05),and there were not signigicant among them(P＞0.05).Conclusions The three shRNA eukaryotic expression plasmids targeted against human TGF-β1(shRNA1,shRNA2,shRNA3)can inhibit the TGF-β1 mRNA expression and protein synthesis in HLF.The shRNA2 has most significant interference effect among them.The growth of HLF transfected with different shRNA from the aspect of morphology and the death rate do not change significantly compared blank group.The shRNA2 can effectively and securely inhibit the expression of TGF-β1 in HLF.