Experimental Study On Paracrine Regulation Of Adipose-derived Mesenchymal Stem Cells On Biological Activities Of Keloid Fibroblasts

Background:Keloid is a fibrotic disease caused by abnormal skin wound healing.It brings heavy physical and mental burden to patients because of its various accompanying symptoms and the characteristics of continuous invasion and growth of similar tumors to the surrounding normal tissues.At present,the clinical treatment methods for keloid can not accurately ensure its curative effect,and is accompanied by a high recurrence rate.New therapies for keloid need to be developed urgently.Recent in vitro and in vivo studies have shown that mesenchymal stem cells(MSCs)have positive therapeutic significance for fibrotic diseases,including keloid.Among them,adipose mesenchymal stem cells(ADSCs)have the most clinical application prospect because they are easy to obtain in large quantities.At present,scholars tend to think that MSC mainly regulates target cells and target tissues through its paracrine pathway,and extracellular vesicles,especially small extracellular vesicles(SEVs)represented by exosomes,are considered to be the main functional carrier of paracrine pathway.However,there is no research to prove that SEVs is the main mediator of ADSCs paracrine regulation of keloid.The research on this problem will further promote the treatment of keloid based on adipose mesenchymal stem cells.Objective:The adipose derived mesenchymal stem cell-conditioned medium(ADSC-CM)of the intervention or non-intervention group of sphingomyelinase inhibitor GW4869 was collected to further isolate and collect the SEVs for characterization analysis,and the conditioned medium of the intervention or non-intervention group was added to the keloid fibroblasts(KFs)or normal fibroblasts(NFs)culture system for further analysis of the biological activities of fibroblasts,including proliferation,migration apoptosis and extracellular matrix protein synthesis,providing a research basis for the role of small extracellular vesicles in the regulation of keloid fibroblasts by paracrine secretion of adipose stem cells.Methods:KFs,NFs and ADSCs were isolated from intraoperative resection or aspiration samples of clinical patients.ADSC-CM obtained from ADSCs with or without adding of sphingomyelinase inhibitor GW4869 was added to the culture system of fibroblasts.The small extracellular vesicles derived from adipose mesenchymal cell regulatory medium were characterized and detected by cryo-electron microscope,nanoparticle tracking analysis and surface marker protein expression analysis.The proliferation,migration,apoptosis and extracellular matrix protein expression of fibroblasts were analyzed by CCK-8 cell proliferation assay,scratch-wound assay,TUNEL staining and western blot.Results:Effects of ADSC-CM on proliferation and proliferation of α-SMA synthesis was inhibitory,it only inhibited the cell migration of KFs but not NFs.ADSC-CM had no effect on fibroblast apoptosis and collagen synthesis.Reducing the content of SEVs in ADSC-CM by neutral sphingomyelinase GW4869 does not affect its regulatory effect on fibroblasts,and GW4869 itself is not cytotoxic to fibroblasts.Conclusions:1.ADSCs can inhibit KFs fibrosis related cell phenotype through paracrine secretion;2.GW4869 can reduce the content of SEVs in ADSC-CM;3.The decrease of SEVs content in ADSC-CM did not affect its inhibition of fibrotic phenotypes of KFs,indicating that SEVs may not be the main functional vectors of ADSCs paracrine secretion...