Effect of the inhibition of smad3 by short hairpin RNA on the expression of smad3 and collagen in keloid fibroblast

Objective:To construct a plasmid generating short hairpin RNA(shRNA)containing the Smad3 gene segment in keloid fibroblasts'(KFB) and to investigate it is effect on the expression of Smad3 and type I collagen(COL1A2) in KFB.Methods:A couple of the most effective siRNA selected from former experiment were used to recombine the plasmids of Smad3 shRNA which were transfected into KFB by Lyo Vec.TM.The expressions of Smad3 and COL1A2 at different time points(0～9d) were detected by RT-PCR and Western blotting.Results:(1) The recombinant Smad3 shRNA vectors were identified by sequence analysis,RT-PCR and Western blotting.(2)The expressions of mRNA and protein of Smad3 in KFB decreased significantly with the extension of time after Smad3 shRNA was transfected and reached the lowest point at 72 hours in experimental group compared with control group by optical density analysis(P0.05).The expressions of mRNA and protein of COL1A2 were also significantly and uniquely decreased following the reduction of Smad3(P0.05).Conclusion:The recombinant Smad3 shRNA vector can suppress the expression of Smad3 in KFB,and decrease the expressions of COL1A2 mRNA and protein.It could be a new promising therapeutic approach to improve skin wound healing and inhibit progression of fibrotic conditions.