| Background Scar is mammalian skin acquired a physiological response to repair wounds,and as one of excessive trauma repair pathological scar forms of keloids(Keloid,K)is a uniquely human,is considered as a fibrosis disease,characterized by excessive Fibroblast(Fibroblast,Fb)proliferation and Extracellular matrix(Extracellular matrix,ECM)which is unusually generated and deposited on the skin dermal tissue,clinical manifestations of hyperplasia of fibrous tissue to wound invasive growth outside the border.So far,the pathogenesis of keloids are still unclear,and lack of effective treatments,serious adverse effects on the physical and mental patients,so the interpretation to the pathogenesis of keloid is still a current hot topic.The existing studies have demonstrated that Mesenchymal Stem Cells(Mesenchymal Stem Cells,MSCs)can relieve the wound inflammation,promote tissue regeneration,and suppress the role of tissue fibrosis,but the rise in the Numbers of MSCs in keloid tissue(keloid-derived Mesenchymal Stem Cells,KD-MSCs)has not been thorough researched.Clinically,keloid with specific direction along the skin tissue tension line attack,the characteristics of international scholars in recent years,studies confirm that holding tension in the different development stages of keloid plays a key role.However,it has not been clarified whether stretch exerts an influence on KD-MSCs and thus participates in the pathogenesis of keloid.Methods1.Isolation,culture and identification of KD-MSCs.Mesenchymal stem cells were isolated by using trypsin,and incubated in stem cell medium.Mesenchymal stem cells in logarithmic phase were choosen and observed under the microscope,then portrayed the growth curve,the experiments such as directionally induced osteogenic and adipogenic multidirectional differentiation and flow cytometry was done in order to appraise the traits of mesenchymal stem cells.2.Isolation,culture and identification of KFs.KFs was separated by tissue mass attachment and cultured with DMEM medium containing 10%FBS.KFs was identified by microscopic observation and immunocytochemistry staining of vimentin.3.Collection of supernatant of KD-MSCs cultured in the stretch group and the unstretched group.With rat tail collagen type ? sidewall and suspended production under tension and no tension group model.The supernatant was collected on 72 h.KFs were cultured with or without KD-MSCs supernatant.After 24 h,48h,the cell morphology was observed under the microscope.In 6 well culture plates keloid fibroblasts were inoculated.After waiting for 24 h,KFs attachment,and KFs were cultured for 48 h in culture medium containing 10% KD-MSCs culture supernatant under stress and no stress respectively,then the morphological changes of cells were observed under microscope.4.Effects of KD-MSCs culture supernatant on KFs proliferation under the stretch group and no stretch group.In 96 well culture plates keloid fibroblasts were inoculated.After waiting for 24 h,KFs attachment,and KFs were cultured for 24 h,48h,72 h,96h in culture medium containing 10% KD-MSCs culture supernatant under stress and no stress respectively.The KFs proliferation was detected by Cell count kits(CCK-8)staining method.5.Under tension and no tension group KD-MSCs culture supernatant on KFs express collagen type ?,type ? and CTGF m RNA.In 6 well culture plates keloid fibroblasts were inoculated,after waiting for 24 h,KFs attachment,and KFs were cultured for 24 h in culture medium containing 10% KD-MSCs culture supernatant under stress and no stress respectively.RNA extraction,c DNA reverse transcription and Real-time PCR detection were done to detect the gene expressions of collagen type ?,type ? and CTGF.6.Under tension and no tension group KD-MSCs culture supernatant on KFs express collagen type?,type ?.In 24 well culture plates keloid fibroblasts were inoculated.After waiting for 24 h,cell attachment,KFs were cultured for 24 h,48h,72 h in culture medium containing 10% KD-MSCs culture supernatant under stress and no stress respectively.The expression of collagen protein type ?,type ? were examined according to the hydroxyproline kit method.Results1.KD-MSCs can directional-induce adipogenic and osteoplastic differentiation,this indicates that KD-MSCs have the characteristics of stem cells.Flow cytometry revealed highly expression of mesenchymal stem cell-associated markers,for instance,the over expression of CD29,CD44 and CD73,and low expressed hematopoietic stem cell surface marker,CD45.The positive rate was highest in CD29.This result indicated that mesenchymal stem cells have keloid phenotypic characteristics of mesenchymal stem cells.2.The time of Fibroblasts primary tissue block culture method was long,the cells which climbed out of the the tissue block were relatively slow.Primary cell boundaries were clear,keloid fibroblasts' arrangements were irregular,and overlapping was visible.With quicker cell proliferation after passage,uniform long shuttle type is the main type under the microscopic,and cell proliferation was active.3.The microscopic main performance for cell in the stretch group and the unstretched group was uniform long spindle,spiral or radial,and growing faster,becoming bulky,gap getting narrow,and contour being darker.Under microscope,there was no significant differences in the effect of KD-MSCs culture supernatant on the morphology of KFs between the effect of tension and non-tension.4.The experimental results of CCK-8 cell proliferation showed that after cultivation for24 h,there was statistical significance in cell number between the groups under stress and no stress(t = 2.76,P < 0.05).However,after 48 h,72 h,96 h,there was no statistical significance between the groups under stress and no stress(t48h = 1.43,t72 h =1.43,t96 h = 0.48,P > 0.05).5.Real-time PCR Experiment results show that comparing the group under stress with the group under no stress,the expression of type ? collagen m RNA increased significantly(t = 14.51,P < 0.01),the expression of type ? collagen and CTGF m RNA decreased(t = 5.20,P < 0.05,t = 0.05,P < 0.01).6.Hydroxyproline colorimetry results showed that comparing the group under stress with the group under no stress,after being cultured with KD-MSCs culture supernatant,the expression of hydroxyproline increased significantly,at 24 h the difference was the most significant,and had statistical significance(t = 4.88,P < 0.05).However,after 48 h,72h,there was no statistical significance between the groups under stress and no stress(t48h = 1.53,t72 h = 3.23,P > 0.05).Conclusion KD-MSCs and KFs were successfully cultured in this study,using rat tail collagen type?sidewall and suspended production tension and no tension group model,and on this basis,it was confirmed that holding tension set of KD-MSCs culture supernatant can promote the KFs proliferation and promote KFs collagen type ? m RNA and protein expression,reduce the KFs collagen type ? m RNA and protein expression,reduce the KFs CTGF expression m RNA.This suggested that stretch can act on KD-MSCs and induce its secretion,and fibroblast proliferation and collagen which was produced by cell factor,and the excessive proliferation of KFs and increased proportion of collagen type?and collagen type ? were characteristics of keloid pathological changes.This suggested that KD-MSCs under stretch may be involved in the pathogenesis of keloid by paracrine action. |
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