The Influence Of Target Regulating Smad4 Of TGF β/Smad Pathway On Hyperplastic Scar Fibroblasts

Purposes:To culture normal fibroblasts and ones derived from hypertrophic scar in vitro and identify them. To detect their differences and investigate siRNA-smad4’s influence on the TGFβ/smad signaling pathway of HS fibroblasts before and after transfection.Methods:Normal fibroblasts and ones derived from pathological scars were cultured in vitro by tissue explants adherent method and identified in terms of their growth characteristics and histochemistry. RNAi technique was employed to achieve oriented expression of silenced smad4 protein in the transfection of LipofectamineTM2000 into fibrocyte. Westernblot and RT-PCR techniques were used to detect smad4, collagenl protein and mRNA expression before and after the transfection of fibroblasts respectively. Furthermore, MTT and FCM were applied to measure the influence of siRNA-smad4 on the proliferative capability, apoptosis and cell cycle of fibroblasts 24,48, and 72 hours post-transfection.Results:Fibroblasts were obtained by culture in vitro. By microscopic, fibroblasts presented spindle growth. In a certain period of time the growth rate of HS fibroblasts was faster than that of normal ones and were identified as Vimentin positive by immunohistochemistry. Smad4mRNA and collagenImRNA expression of hypertrophic scars is strengthened compared to normal skin fibroblasts (P<0.05) and was reduced after transfection (P<0.05). The proliferation ability of HS fibroblasts was significantly suppressed after transfection. Cell cycle was greatly shortened, which promoted the apoptosis of cells.Conclusion:Smad4 protein and collagenI expressions were reduced through siRNA-smad4’s target regulating TGFβ/smad signaling pathway. Synthesis of extracellular matrix was reduced to some extent. Proliferation of hibroblasts was inhibited and apoptosis was induced. An effective target for gene therapy was provided for the prevention and treatment of pathological scars.