The Expression Of DNMT1in Keloid Fibroblasts And The Effect Of5-AZA-2-deoxycytidine On Keloid Fibroblasts

CHAPTER1EXPRESSION AND SIGNIFICANCE OFDNMT1IN HUMAN KELOID FIBROBLASTObjective: To investigate the expression and significance ofmethylation specific enzyme1(DNMT1) in different parts and formationtime of keloid fibroblasts.Methods: Fifty-three patients with keloid surgical excision of keloidswere grouped according to formation time; Immunohistochemistry wasused to assay the expression of DNMT1in normal skin, invasive part,proliferative part and aged part; RT-PCR was adopted to assay theexpression of DNMT1mRNA in each group.Results: DNMT1are mainly distributed in the nucleus of fibroblasts;the expression of DNMT1protein and DNMT1mRNA in keloid werehigher than those of normal skin fibroblasts(p<0.05); The expression rate ofDNMT1protein and DNMT1mRNA in shorter formation time group werehigher than in longer formation time group(p<0.05); The expression rate of DNMT1protein and DNMT1mRNA in the invasive part of keloid werehigher than in proliferative part and aged part.Conclusion: DNMT1played an important role in keloid; Theexpression of DNMT1tended to decrease along with the increase of theformation time in keloid,It also related to the part of keloid. DNMT1played an important role in the process of keloid growth and closely relatedto infiltrative growth in keloid.CHAPTER2KELOID ESTABLISHMENT ANDGROWTH CHARATERISTICS OF THE FIBROBLASTCELL LINESObjective: Stable method of keloid fibroblasts primary culturedfibroblasts cultured in vitro growth characteristics, and preliminaryreseacrch into buiding a solid foundation for the later stages of theexperiment.Methods: A single-cell method in primary cultured keloid fibroblasts,inverted optical microscope fibroblast growth morphology was observed byMTT assay vitro growth curve of keloid fibroblasts.Result: Keloid fibroblasts growthed to adherent about3days andsuceessfully passaged about15days, fibroblast cells were fusiform or starpolarity which arranged radially or swirling, MTT assay measured that keloid fibroblasts growthed curve presents the “S” type, the cells growthedwell and suitable for further experiments.Conclusin: We completed keloid fibroblast cells culture successfullyin vitro, keloid fibroblasts growthed in good condition and providedmaterial for next experiment.CHAPTER3EFFECT OF5-AZA-2-DEOXYCYTIDINEON CELL CYCLE AND APOPTOSIS OF HUMAN KELOIDFIBROBLASTSObjective: To investigate the inhibition effect of methylase inhibitor5-aza-2-deoxycytidine on human keloid fibroblasts, and influence ofcytokines on human keloid fibroblasts.Methods: Human keloid and surrounding normal skin samples werecollected for primary cultured fibroblasts, The cells were divided intokeloid fibroblasts experimental group, normal skin fibroblasts experimentalgroup, keloid fibroblasts control group and normal skin fibroblasts controlgroup.5-Aza-2-deoxycytidine was used to intervented the experimentalgroups. The changes of cell cycle and apoptosis of fibroblasts whicheffected by5-Aza-2-deoxycytidine were analysed with flowcytometry(FCM).Results: Compared with normal skin fibroblasts, The proportion of cells in G0/G1stage(P=0.035)and apoptosis cells(P=0.006)were less thanthe keloid fibroblasts; The FCM showed that the proportion of cells inG0/G1stage was increased(P=0.000), and so was the proportion ofapoptosis cells(P=0.047)in keloid fibroblasts intervented by5-aza-2-deoxycytidine. But the change of normal skin fibroblast group wasinsignificant(P>0.05).Conclusion: Methylaze inhibitors,5-aza-2-deoxycytidine, may inhibitthe proliferation and promote apoptosis of keloid fibroblasts. But the effecton normal skin fibroblasts was insignificant. The pathogenesis of keloidmay be related to the methylation of certain genes.5-aza-2-deoxycytidinemay be a new choice for the treatment of pathological scar.CHAPTER4EFFECT OF5-AZA-2-DEOXYCYTIDINE ONRELATED CYTOKINES OF HUMAN KELOIDFIBROBLASTSObjective: To investigate the inhibition effect of methylase inhibitor5-aza-2-deoxycytidine on human keloid fibroblasts.Methods: Human keloid and surrounding normal skin samples werecollected for primary cultured fibroblasts, The cells were divided intokeloid fibroblasts experimental group, normal skin fibroblasts experimentalgroup, keloid fibroblasts control group and normal skin fibroblasts control group,5-Aza-2-deoxycytidine was used to intervented the experimentalgroups. The expression of DNMT1, TGF-β1and Smad7mRNA in eachgroup were detected with RT-PCR.Results: Compared with normal skin fibroblasts, The expression ofDNMT1and TGF-β1mRNA was more(P=0.001)but Smad7mRNA wasless(P=0.001); The expressions of DNMT1and TGF-β1mRNAdecreased(P=0.003)but that of Smad7mRNA elevated(P=0.000)in keloidfibroblasts intervented by5-aza-2-deoxycytidine. But the change of normalskin fibroblast group was insignificant(P>0.05).Conclusion: Methylaze inhibitors,5-aza-2-deoxycytidine, mayinfluence the expression of related cytokines in keloid fibroblasts. But theeffect on normal skin fibroblasts was insignificant. The pathogenesis ofkeloid may be related to the methylation of certain genes.5-aza-2-deoxycytidine may be a new choice for the treatment of pathological scar.