The Expression Of ShRNA Suppressing Smad3 In KFB

PART ONE The construction of shRNA-Smad3 vector and cultivation of primary human keloid-derived fibroblast cellObjective: To construct a short hairpin RNA(shRNA) vector suppressing Smad3 gene of keloid fibroblasts'(KFB) expression in order to investigate the effect of parameters.Methods: A couple of the most effective siRNA selected from former experiment were recombined as expression plasmids of Smad3 shRNA to transfect into KFB by Lyo Vec.TM And choose different DNA dose, liposome concentration and transfection duration. The expression of shRNA-Smad3 and cell survival condition were observed by fluorescence microscope at the different time-periods after transfection.Results: Rank analyzing and gelose electrophoresis confirm succession constructed of shRNA-Smad3. Fibroblast was transfected for 6 hours without blood serum.The transfection was obvious 72 hours after transfection when the plasmid DNA was added to liposome in the proportion of 1:1.8.Conclusion: On the condition of proper liposome concentration, fairly short transfection duration, the primary cultural human keloid-derived fibroblast transfected by the method mediated by liposome can make destination gene obtain higher transfection efficiency and the least cell death. So the method mediated by liposome is a fairly ideal transfection way to research biological behaviour of the primary cultural human keloid-derived fibroblasts. The primary human keloid-derived fibroblast can been cultural by tissuePART TWO Effection of Smad7,Smad3 and COL1A2 gene expressed in KFB after Smad3 shRNAObjective:To investigate it's effect on the expression of Smad7,Smad3 and COL1A2 in KFB after Smad3 shRNA have been transfected into KFB by Lyo Vec.TM.Methods: A couple of the most effective siRNA selected from former experiment were recombined as expression plasmids of Smad3 shRNA to transfect into KFB by Lyo Vec.TM The expressions of Smad3,Smad7 and COL1A2 at different time point (0～9d) were detected by RT- PCR and Western blotting.Results:①The recombinantion of Smad3 shRNA vector was identified by RT-PCR and Western blotting.②After Smad3 shRNA be(was) transfected, the expression of mRNA and protein of Smad3 in KFB of experiment treated group decreased significantly with the extension of time and reached the lowest point at 72 hours, the differences were statistically significantly by optical density analysis between experiment treated group and control group (P<0.05).In contrast, the expression of mRNA and protein in Smad7 increased significantly(P<0.05).Conclusion: The eukaryotic expression vector of Smad3 shRNA constructed in vitro could suppress the expression of Smad3 in KFB,but increased Smad7 mRNA and protein rapidly. The change suggest that Smad3 may function as a reverse regulation(or) of Smad7.