Specific ShRNA Targeting Of COX-2 Influenced α-SMA,Collagen 1,TGF-β₁ In Rat Hepatic Stellate Cells

ObjectiveResearch the expression of COX-2,α-smooth muscle actin (α-SMA), collagen type 1, transforming growth factor-β₁ (TGF-β₁) in HSC by the way of COX-2shRNA .Methods(1)Transfer three different COX-2shRNA vector into HCS by Lipofectamine2000, observe the transfection efficiency by fluorescence microscope, RT-PCR and western blot verified vectors.(2)After 24h, 48h and 72h by transfer COX-2shRNA, RT-PCR measure the expression of COX-2,α-SMA, collagen type 1 and TGF-β₁ in HSC by the way of COX-2shRNA and nimesulide.Results(1) immunofluorescence assay show: transfer COX-2shRNA vector into HCS,we can see green fluorescence cytoplasm in HSC by fluorescence microscope. The transfection efficiency of COX-2shRNA-1,COX-2shRNA-2 and COX-2shRNA-3 expression vector are over 70%.(2) The gene silence of COX-2: RT-PCR show After 48h by transfer COX-2shRNA,the mRNA of COX-2 are inhibit by three different COX-2shRNA vector(p<0.05),the best inhibitory effect is COX-2shRNA-1.(3) The expression of COX-2 by different treatment method in HSC: after 24h by transfer COX-2shRNA, the expression of COX-2 in COX-2shRNA-1 and nimesulide were lower than control group and empty vector group(p<0.05); after 48h and 72h by transfer COX-2shRNA, the expression of COX-2 in three different COX-2shRNA and nimesulide were lower than control group and empty vector group(p<0.05),the best effect is COX-2shRNA-1 transfer HSC after 72h.(4) The expression ofα-SMA by different treatment method in HSC: after 24h by transfer COX-2shRNA, the expression ofα-SMA have no statistical significance in different group; after 48h by transfer COX-2shRNA, the expression ofα-SMA in COX-2shRNA-1, COX-2shRNA-2 and nimesulide were lower than control group and empty vector group(p<0.05); after 72h by transfer COX-2shRNA, the expression ofα-SMA in three different COX-2shRNA and nimesulide were lower than control group and empty vector group(p<0.05),the best effect is COX-2shRNA-1 transfer HSC after 72h.(5) The expression of collagen type 1 by different treatment method in HSC: after 24h,48h and 72h by transfer COX-2shRNA, the expression of collagen type 1 in COX-2shRNA-1 and nimesulide were lower than control group and empty vector group(p<0.05),the best effect is COX-2shRNA-1 transfer HSC after 72h.(6) The expression of TGF-β₁ by different treatment method in HSC: after 24h,48h and 72h by transfer COX-2shRNA, the expression of TGF-β₁ in three different COX-2shRNA and nimesulide were lower than control group and empty vector group(p<0.05),the best effect is COX-2shRNA-1 transfer HSC after 72h.Conclusions(1) The gene COX-2 can be silence by COX-2shRNA transfer HSC.(2) COX-2shRNA can decrease the proliferation of HSC, decrease the collagen and TGF-β₁ by silence COX-2.