Influence Of RNAi Silencing Smad3 On The Functions Of Keloid Fibroblasts

PART ONE The influence of SiRNA-Smad3 on smad3 gene expression of keloid fibroblastObjective: To probe the influence of SiRNA-Smad3 on smad3 gene expression of keloid fibroblast.Methods: Three pairs of chemically synthesized SiRNA-Smad3 (Y1,Y2,Y3)were adopted to transfect the cultured human keloid fibroblast (KFB ) respectively by the interfering technology of micromolecule RNA with carrier of positive ion liposome, while KFBs without transfection of SiRNA-Smad3 were used as control to interfere the expression of Smad3 and block signal transduction of endogenous TGF-β/Smads; Expression of Smad3 was detected using RT-polymerase chain reaction (RT-PCR) and Western blot after interfering with SiRNA-Smad3. The change of distribution of Smad3 expression in KFB before and after SiRNA-Smad3's interference was examined by immunofluorescence chemical assay.Results: Expression of Smad3 mRNA and protein in KFB was interfered by RNAi technology. The expression of Smad3 mRNA determined by RT-PCR was weakest in group Y1 of the three, which was coincident with RT-PCR while using Western Blot. The interference of SiRNA-Smad3 could decrease the expression of Smad3 in cytoplasm and reduce its transfer from cytoplasm to cell nucleus obviously.Conclusion: Expression of Smad3 mRNA and protein was inhibited in human KFB transfected by SiRNA-Smad3 specifically, which resulted in less expression of Smad3 in cytoplasm and nearly no expression in cell nucleus obviously.PART TWO The influence of SiRNA-Smad3 silencing Smad3 on proliferation and apoptosis of keloid fibroblastsObjective: To research the regulating effects of SiRNA-Smad3 silencing Smad3 on proliferation and apoptosis of keloid fibroblasts (KFB).Methods: SiRNA-Smad3 was used to transfect the primary cultured human keloid fibroblasts by liposome method. The proliferative behavior of KFB was studied by 4 methyl thiazolyl tetrazolium method under the endogenous TGF-β/Smads signal transduction blocked. The cell cycle and apoptosis were determined by flow cytometry under the two conditions.Results: Not only was the proliferation of KFB inhibited, but also apoptosis was induced with endogenous TGF-β/Smads signal transduction blocking. Conclusion: TGF-β/Smads signal may regulate the proliferation and apoptosis of KFB at a certain concentration.PART THREE The influence of SiRNA-Smad3 silencing Smad3 on expression of FN in the human keloid fibroblastObjective: To study the regulating effects of TGF-β/Smads signal on expression of FN in the human keloid fibroblasts (KFB).Methods: SiRNA-Smad3 was used to transfect the primary cultured human keloid fibroblasts by liposome method. Expression of FN in KFB was detected by RT-PCR, Western Blot and immunohistochemistry under the condition of endogenous TGF-β/Smads signal transduction blocked.Results: The expression of FN in keloid fibroblast was down-regulated when endogenous TGF-β/Smads signal transduction has been blocked. Conclusion: Smad3 may inhibit expression of FN in keloid fibroblasts by mediating TGF-β1 signal.PART FOUR The influence of T SiRNA-Smad3 silencing Smad3 on expression of ColⅠin the human keloid fibroblastObjective: To study the regulating effects of TGF-β/Smads signal on expression of ColⅠin the human keloid fibroblasts (KFB).Methods: SiRNA-Smad3 was used to transfect the primary cultured human keloid fibroblasts by liposome method. Expression condition of ColⅠin KFB was detected by RT-PCR,Western Blot and immunohistochemistry on condition that endogenous TGF-β/Smads signal transduction has been blocked.Results: The expression of ColⅠin keloid fibroblast was attenuated when endogenous TGF-β/Smads signal transduction was blocked.Conclusion: Smad3 may down-regulate expression of ColⅠin keloid fibroblasts by mediating TGF-β1 signal.