Abstract： Objective To investigate the effect of Gsh2 silencing on the sonic hedgehog (Shh) and GLI1 gene expression,which are the key factors of hedgehog pathway in human pancreatic cancer cell SW1990.Methods Three shRNAs tagerting Gsh2 (Gsh2-shRNA) were constructed and were inserted into pLKO.1 puro vector by double digestion with restriction enzyme,the empty vector without shRNA was used as a negative control.After plasmid amplification,plasmids DNA was extracted,and underwent electrophoretic separation and sequencing by double digestion.After confirmation by gene sequencing,plasmid was transfected into the human pancreatic cancer cell line SW1990,then real time PCR was used to detect the Gsh2 mRNA expression,and the plasmid with best silencing effect was selected to transfect the SW1990 cells,real time PCR and Western blot were respectively used to detect the mRNA and protein expression of the key factor in hedgehog pathway(Gsh2,Shh,Gli1).Results The gene sequencing of the 3 recombinant plasmids showed that Gsh2-shRNA were correctly constructed.If the mRNA expression of empty vector transfection cells was 1,the Gsh 2 mRNA expressions of SW1990 cells which were transfected by recombinant Gsh2-shRNA-1,Gsh2shRNA-2,Gsh2-shRNA-3 were 0.41 ± 0.02,0.22 ± 0.043,0.53 ± 0.03,and Gsh2-shRNA-2 had the best silencing effect.If the mRNA or protein expression of empty vector transfection cells was 1,the Gsh2,Shh,Gli1 mRNA expressions of cells with Gsh2-shRNA-2 transfection were 0.12 ±0.02,0.97± 0.13,0.19± 0.03,and the protein expressions were 0.55 ± 0.12,0.74 ± 0.06,0.53 ± 0.09.The Gsh2,Glil mRNA and protein expressions with Gsh2-shRNA-2 transfection were significantly down-regulated when compared with those of empty vector transfection cells,and the difference between the two groups was statistically significant (P＜0.01 or ＜0.05).Conclusions Gsh2 gene silence in pancreatic cancer cell line SW1990 can repress the expression of GLI1 in hedgehog pathway without affecting Shh expression.