Effect Of The Methylation Enzyme Inhibitors Of5-aza-2Deoxycytidine On The TGF-B/smad Signal Transduction Pathway In Human Keloid Fibroblasts

chapter1the protein expression of phosphorylation smad2(p-smad2),phosphorylationsmad3(p-smad3) in keloids andnormal skinObjective:Immunohistochemical SP was used to research proteinexpression and differences of phosphorylation smad2(P-smad2),phosphorylation smad3(P-smad3) in keloids and normal skin tissue.Method:Collection of48cases keloids and other cosmetic surgerypatients of normal skin tissues. Immunohistochemical SP-AP method todetect the distribution of P-smad2and P-smad3protein expression in lowpower and high power microscope respectively.Results:The protein positive expression rate of P-smad2and P-smad3inkeloid was higher than in normal skin tissue (P <0.05).Conclusion:The protein expression differences of P-smad2and P-smad3in keloid and normal skin suggest that they may participate in keloidformation,providing certain theroretical basis for the drug therapy. Chapter2keloid and normal skin fibroblasts were cultivated invitro and we studied the effect of5-aza-2deoxycytidine on cellgrowth cycle and apoptosis in keloid and normal skin fibroblastsObjective:To develop enough keloid and normal skin fibroblasts lay thefoundation for experiment, methylation inhibitors5-aza-2deoxycytidineintervention, cell cycle and apoptosis of keloid fibroblasts were anaylzedby flow cytometry instrument.Methods:Using tissue block method to cultivate of keloid and normal skinfibroblasts, methylation inhibitors5-aza-2deoxycytidine intervention, cellcycle and apoptosis of keloid fibroblasts were analyzed by flow cytometryinstrument.Results: Primitive cells using tissue explant culture climbed out of the cellsin about7days, about14days after the fibroblast covered the entire culturebottle.Flow cytometry instrument shown after5-aza-2deoxycytidine wasused to intervent keloid fibroblasts,the rate of cells stagnation in G0/G1phase and apoptosis were increased, while the normal skin fibroblasts hadno obvious changed.Conclusion:5-aza-2deoxycytidine could increase the rate of cellsstagnation in G0/G1phase and apoptosis in keloid fibroblasts.It instructedthat5-aza-2deoxycytidine can inhibit proliferation and promote apoptosisof keloid fibroblasts. Chapter3The effect of5-aza-2-deoxycytidine on keloidfibroblasts related factorsObjective:Research related factor expression changes of TGF-β／Smadsignal transduction pathways after5-Aza-2-CdR interventing in keloids andnormal skin fibroblastsMethods:RT-PCR was made to detect and compare collagen (Coll) ⅠmRNA expression changes of keloid and normal skin fibroblasts and afterdrug intervention. Western blot analyzed protein expression changes of P-smad2, P-smad3, TGF-beta1, smad7and Coll-Ⅰ.Results:The expressions of Coll-ⅠmRNA was decreased in keloidfibroblasts,the protein expression of P-smad2, P-smad3, TGF-β1,Coll-Ⅰwere significantly suppressed while expression of smad7increasedwith5-Aza-2-CdR. However,5-Aza-2-CdR did not influence TGF-β/smadsignaling pathway in normal skin fibroblasts.Conclusion:We conclude that the5-aza-2-deoxycytidine inhibits themethylation through the inhibition of DNMT1expression, then the level ofSmad7rebound from its original lowered level,which makingphosphorylated smad2and smad3decline.After that, the essentialTGF-β/smads pathway may be restrained.As results,collagen synthesismight be reduced.