Study On The Effects Of Bone Marrow Derived Mesenchymal Stem Cells On The Expression Of Methylation Specific Enzyme 1 In Keloid Fibroblasts

BackgroundKeloid is a special type of scar,which is a hyperplastic skin disease,fibrotic tumors caused by a collection of atypical fibroblasts with excessive deposition of extrace llular matrix components,especially collagen,fibronectin,elastin,and proteoglycans.The formation of keloids have not yet been well established and physicians cannot(or at least find it very difficult to)control systemic and genetic risk factors of keloids and hypertrophic scars.However,they can use a number of treatment modalities that all,prophylactic and treatment strategies remain unsatisfactory.The recent research found that the expression of DNMT1 in keloid were higher than those of normal skin fibroblasts,DNMT1 played an important role in keloid.Methylation inhibitors may inhibit the methylation through the inhibition of DNMT1 expression,then decreased expression of TGF-β 1,P-smad2,P-smad3,and the essential TGF-β/smads pathway maybe restrained.Researcher have reported that BMSC-conditioned medium inhabited KFs proliferation and migration,BMSC-conditioned medium significantly decreased expression of TGF-β and suppressed the ECM synthesis in KFs.So we can guess whether BMSCs microenvironment could inhibit the expression of DNMT1 in co-cultured keloid fibroblasts,thus inhibiting the keloid fibroblasts by regulating TGF-β/Smad signal transduction pathway.We established indirect co-culture system using Transwell chamber.The expression changes of DNMT1 were detected by immunofluorescence,Western Blot and real-time fluorescent PCR.Western Blot assay was used to detect the protein expression of TGF-β1.RT-PCR was used to detect the expression of Smad7.To investigate the effects of BMSCs on the expression of DNMT1 and related factors expression changes of TGF-β/Smad signal transduction pathways in KFs.PART Ⅰ CULTURE,IDENTIFICATION OF THE BMSCS,NHDFS,KFS,AND ESTABLISH A CO-CULTURE SYSTEMObjective:To study the effects of BMSCs on fibroblasts,we need to culture and identify in vitro of BMSCs and fibroblasts,and establish an indirect co-culture system using Transwell chamber.Methods:1)The Human keloid fibroblasts and human normal skin fibroblasts were identified by HE staining and immunocytochemistry;2)expression of CD29,CD44 and CD45 were assayed by flow cytometry;3)Using Transwell chamber to establish the co-culture system.The morphological changes of the fibroblasts were observed by invert microscope.Results:1)Hematoxylin-eosin staining showed that the cells were clostridial form or irreguler,with pale blue nuclei and pink cytoplasm,and immunocytochemical staining with vimentin showed that the cultured cells were vimentin positive,and the positive results were tan at the antigen location.2)Microscopy showed that BMSCs with spindle and fibroblast-like shape,CD29 and CD44 showed positive expression,CD45 showednegative exoression in the BMSCs by FLC;3)The co-culture system was successfully established by using Transwell chamber.After co-cultured with BMSCs,KFs decreased significantly and was spirally arranged.Conclusion:The indirect co-culture system could be established by Transwell chamber,BMSCs microenvironment had an effect on the morphological characteristics of the KFs.PART Ⅱ THE EFFECTS OF BMSCS MICROENVIRONMENT ON DNMT1 OF KELOIDSObjective : To study the microenvironment constituted of BMSCs paracrine factors on DNMT1 of KFs.Methods : In this study,fibroblasts were divided into four groups:NHDFs group,co-cultured NHDFs group,KFs group,co-cultured KFs group.The expression changes of DNMT1 were detected by immunofluorescence,Western Blot and real-time fluorescent PCR after 48 hours.Results : Immunohistochemistry experiments showed that DNMT1 showed high expression in KFs.Western blot and RT-PCR results showed that the expression of DNMT1 in co-cultured KFs group was significantly reduced than that in the control group(P =0.000).There was no significant difference in protein and m RNA expression of DNMT1 between the co-cultured NHDFs group and the NHDFs group.Conclusion : The results of this section showed that Bone marrow derived mesenchymal stem cells microenvironment could reduce the expression of DNMT1 of KFs.PART Ⅲ THE EFFECTS OF BMSCS MICROENVIRONMENT ON TGF-Β/SMAD SIGNAL TRANSDUCTION PATHWAYS IN KFSObjective:To investigate the effects of BMSCs on the expression of related factor expression changes of TGF-β/Smad signal transduction pathways in KFs.Methods : In this study,fibroblasts were divided into four groups:NHDFs group,co-cultured NHDFs group,KFs group,co-cultured KFs group.Western Blot assay was used to detect the protein expression of P-smad2、P-smad3、TGF-β1,MMP2.RT-PCR was used to detect the expression of Smad7.Results : After co-cultured with BMSCs,the protein expression of P-smad2、P-smad3、TGF-β1 in KFs was significantly reduced(P =0.000)and Smad7 m RNA expression of KFs group was increased(P=0.002).while reducing MMP-2 production.Conclusion:We conclude that the BMSCs microenvironment inhibits the expression of DNMT1 in KFs,then the essential TGF-β/smads pathway may be regulated,while decreasing synthesis of extracellular matrix.