Primary Study On Epigenetics And Pathogenesis Of Pathological Scar

CHAPTER 1:EXPRESSION AND DIFFERENCES OF MECP2 IN PATHOLOGICAL SCAR FIBROBLASTSObjective: To investigate the expression and differences of MECP2 in fibroblasts of human normal skin,normal scar,keloid and hypertrophic scar at different growth stages.Methods: The specimens of clinical patients are collected,33 cases of normal skin,32 cases of normal scar,51 cases of hypertrophic scar and 35 cases of keloid;and then the hypertrophic scar are divided into four groups according to the growth time: 0-3M,3-6M,6-12 M,> 12 M.Then,the expression levels of MECP2 protein and mRNA in fibroblasts were detected by immunohistochemistry,Western Blot and qRT-PCR.Results: Compared with normal skin,the expressions of MECP2 protein and mRNA in normal scar,hypertrophic scar and keloid tissue were significantly different and gradually increased.MECP2 expression was also different when hypertrophic scars at different growth stages;there was no significant difference in MECP2 expression when hypertrophic scars at the early stage(0-3M)and the proliferative stage(3-6M),but MECP2 expression was significantly reduced when hypertrophic scars at the stable stage(6-12M)and the fading stage(> 12M).Conclusion: MECP2 plays a crucial role in pathologic scar formation.MECP2's differences among the groups suggest that :the representation of MECP2 is directly related to the degree of fibrosis,which provides a new direction to inhibit the formation of pathologic scar.CHAPTER 2:EXPRESSION AND DIFFERENCES OF HDAC6 IN PATHOLOGICAL SCAR FIBROBLASTSObjective: To investigate the expression and differences of HDAC6 in fibroblasts of human normal skin,normal scar,keloid and hypertrophic scar at different growth stages.Methods: The specimens of clinical patients are collected,33 cases of normal skin,32 cases of normal scar,51 cases of hypertrophic scar and 35 cases of keloid;and then the hypertrophic scar are divided into four groups according to the growth time: 0-3M,3-6M,6-12 M,> 12 M.Then,the expression levels of MECP2 protein and mRNA in fibroblasts were detected by Western Blot and qRT-PCR.Results: The expression of HDAC6 in keloid was significantly higher than that in normal skin,while the expression of HDAC6 in hypertrophic scar was between the normal sacr and keloid,and HDAC6 in normal skin was the lowest.HDAC6 expression was also different in hypertrophic scar at different growth times.There was no significant difference when hypertrophic scar at the early stage(0-3M)and the proliferative stage(3-6M),but HDAC6 expression was significantly reduced when hypertrophic scar at the stable stage(6-12m)and the fading stage(> 12M).Conclusion: Histone deacetylation plays an important role in the process of pathological scar occurrence.The high expression of HDAC6 in keloid indicates that the epigenetic regulation of histone deacetylation is a new research target.CHAPTER 3: ANALYSIS OF METHYLATION STATUS OF SMAD7 GENE PROMOTER IN PATHOLOGICAL SCAR FIBROBLASTSObjective: To investigate the methylation status of Smad7 gene promoter in normal skin and pathological scar.Methods: Total DNA of normal skin and pathological scar was extracted by centrifugal column method,and then DNA was modified by bisulfite,MSP technology was used to detect the methylation status of Smad7 gene promoter.Results: The methylation of Smad7 gene promoter in keloid group was significantly higher than that in other groups.Conclusion: Hypermethylation of Smad7 provides a precursor for MECP2 binding,and the relationship between MECP2 and Smad7 in pathologic scar can be further studied.CHAPTER 4:EFFECT OF MECP2 EXPRESSION INHIBITION ON KELOID FIBROBLASTSObjective:To investigate the proliferation ability of human keloid fibroblasts after MECP2 was inhibited,and whether the expression of Samd7,TGF-1,?-SMA and p-Smad2 were altered.Methods: Human keloid fibroblasts were cultured routinely.SiRNA-MECP2 was transfected into keloid fibroblasts by RNA interference technique,and its transfection efficiency was verified by qRT-PCR and Westren Blot.Then,the proliferation ability of fibroblasts at0 h,24h,48 h and 72 h was detected by MTT technique,and the changes of Samd7,TGF-1,?-SMA,p-Smad2 protein and mRNA in keloid fibroblasts were detected by Western Blot and qRT-PCR.Results: After SiRNA-MECP2 was successfully transfected into human keloid fibroblasts,the proliferation ability of keloid fibroblasts gradually decreased with the extension of the transfection time.Smad7 protein expression was increased after MECP2 expression was inhibited and the expression of cytokines TGF-1,?-SMA,p-Smad2 protein and mRNA related to cell fibrosis were decreased.Conclusion:After the expression of MECP2 was inhibited in keloid fibroblasts,the cell proliferation and corresponding cytokines were changed,which indicating that MECP2 not only significantly inhibits theexpression of Smad7,but also promotes the fibrosis of pathological scars.