Effect Of Human Fetal Dermal Mesenchymal Stem Cells On Bioactivities Of Keloid Fibroblasts

BackgroundKeloid is one kind of benign skin diseases caused by hyperplasia of fibroblasts and collagen fibrils.The present study has not revealed its etiology and growth mechanism due to the complexity of its formation mechanism.It is refractory due to lacking effective treatments at present,which put pressure on seeking new therapeutic regimen.Mesenchymal stem cells(MSCs)have arisen in the clinical treatment of fibrosis diseases because of their anti fibrosis effect.Fetal dermal mesenchymal stem cells(FDMSCs)are considered to play a crucial role in scarless healing.Nevertheless,the efficacy of them on keloid remains poorly understood.ObjectiveTo observe effects of FDMSCs conditioned medium(F-CM)on keloid fibroblasts(KFs)in vitro and effects of cells on bioactivities of KFs in vivo,then to explore the feasibility for preventing and treating keloid through related FDMSCs therapy.MethodsAdult dermal fibroblasts(ADFs),FDMSCs and KFs were isolated to single cells and cultured in DMEM.ADFs and FDMSCs were used to generate ADFs conditioned medium(A-CM)and F-CM.KFs were cultured with A-CM and F-CM in vitro compared with control group.After 48h incubation,a series of experimental analysis is given based on KFs,including cells morphology(observed by invert microscope),cell number varied(MTT assay),The rates of cell proliferation and apoptosis(BrdU cell proliferation assay and TUNEL cell apoptosis assay),apoptosis-associated genes and proteins(qPCR and Western blot),stage of cells apoptosis(annexin V-FITC/PI binding assay).ResultsCompared with adult group and control group,F-CM could change the morphology of KFs to typical apoptosis morphology and decrease the number of living KFs.A-CM and F-CM had no significant effect on KFs proliferation while the percentage of TUNEL-positive cells of F-CM group was much higher than control group and A-CM group,suggested that F-CM reduced the number of living KFs by inducing cell apoptosis mainly.Consistent with the results of conditioned medium intervention experiment,co-culture of FDMSCs and KFs also reduced the number of living KFs by inducing cell apoptosis mainly.F-CM promoted expression of pro-apoptotic proteins and inhibited expression of anti-apoptotic proteins to accelerate the apoptosis.Furthermore,F-CM accelerated the late phase of KFs apoptosis mainly.Conclusion(1)FDMSCs can be successfully isolated from fetal skin and cultured for long periods,with maintenance of biological characteristics,which meet the needs of clinical application.(2)The results of conditioned medium group were consistent with the results of cell co-culture group,which avoided direct application of the stem cells on the human.(3)F-CM promoted expression of pro-apoptotic proteins and inhibited expression of anti-apoptotic proteins to accelerate the apoptosis.(4)F-CM accelerated the late phase of KFs apoptosis mainly.