Study On The Mechanism And Effects Of Secreted Factors Of Adipose Derived Mesenchymal Stem Cell On Fibrosis Phenotype Of Pathological Scar Fibroblasts

BackgroundHypertrophic scars and keloids are fibrotic diseases caused by dysregulation of the wound healing processes,with excessive proliferation of fibroblasts,epithelial-mesenchymal transformation,increased myofibroblasts and extracellular matrix(ECM)deposition.Excessive ECM deposition is the main pathological feature.Histological differences between the two are not obvious,but as to clinical manifestations aresignificant,hypertrophic scars are often confined to the site of injury,higher than the skin surface,with occasional pain,itching and other discomfort,the development period often less than a year,and usually is self-limited.While keloids are often invasive,extend to the normal skin around the lesion,significantly higher than the surface of the skin,also with pain and itchiness,the development period can be up to several years.The complex pathophysiological mechanism of these diseases results in the lack of effective treatment methods.In recent years,stem cells have been widely used in plastic and reconstructive surgery.A large number of studies have shown that mesenchymal stem cells(MSCs)can promote high-quality tissue healing.Compare with other kinds of MSCs,the human adipose-derived mesenchymal stem cells(hADSCs)with advantages of abundant resources,minimal invasion,and better cell viability.However,there is no related study detected effects of adipose derived mesenchymal stem cells on hypertrophic scars and fibroblasts derived from keloids.A great body of studies have shown that TGF-?1 activation is the classic driving force for skin fibrosis,and the TGF-?1/Smad pathway is a classic pathway for fibrotic diseases.Inhibition of this signaling pathway can effectively block the progression of fibrosis.The Notch signaling pathway was found to cooperating with TGF-?1 in renal fibrosis,pulmonary fibrosis,and hepatic fibrosis,promoting the progression of fibrosis,and Notch signaling was also found to be highly expressed in skin scar tissue,especially in keloids.ObjectiveTo investigate the paracrine effects of human adipose derived MSCs(hADSCs)on the biological behavior and fibrosis phenotypes of hypertrophic scar fibroblasts(HSFs)and keloid fibroblasts(KFs).To provide the experimental evidence for the mechanism and clinic application of hADSCs conditioned medium on attenuating the formation of hypertrophic scar and keloid.Methods1.Adipose tissue specimens from liposuction were obtained,and human ADSCs were isolated and cultured.hADSCs of passage 3 were expanded and cultured for 12h,24h,and 48h to obtain 12h hADSCs conditioned medium(12h-CM),24h hADSCs conditioned medium.(24h-CM),48h hADSCs conditioned medium(48h-CM),low-sugar DMEM without FBS were incubated in the incubator Oh,12h,24h,48h to obtain no-treatment control group medium(NC)and 12h,24h 48h condition control media(12h-CC,24h-CC,48h-CC).2.Obtained normal human skin,hypertrophic scars and keloid specimens,were cutted into a 4 ?m thick tissue section for immunohistochemical staining,the remaining specimens were isolated and cultured to get NFs,HSFs,and KFs,and passage 3 were storage at liquid nitrogen containers for future detecting.3.The NFs,HSFs,and KFs were treated with NC,condition control media,and 12h-CM,24h-CM,and 48h-CM for 24 hours separately.Cell viability were detected by CCK-8 method,respectively.Wound healing was used to detect migration ability,MUSE kit was used to detect apoptosis and distribution of cell cycle,and LDH kit was used to detect cytotoxicity.4.The NFs,HSFs,and KFs were treated with NC,condition control media,and 12h-CM,24h-CM,and 48h-CM for 24 hours separately.The expression of fibrosis-related genes was detected by real-time PCR.5.The NFs,HSFs,and KFs were treated with NC,condition control media,and 12h-CM,24h-CM,and 48h-CM for 24 hours separately.Real-time PCR was used to detect expression of extracellular matrix components,such as type I collagen and fibronectin at the mRNA level.The content of hydroxyproline in the cell culture supernatant was detected to detect the synthesis and secretion of extracellular matrix.6.Tissue immunohistochemical and cell immunofluorescence staining was used to detect the expression of a-SMA in keloid,hypertrophic scar and normal skin.The NFs,HSFs and KFs were treated with NC,condition control media and 12h-CM,24h-CM,and 48h-CM for 24 hours.Real-time PCR was used to detect Notch 1,Jagged-1(JAG).-1,Notch ligand),Jagged-2(JAG-2),Delta-1,TGF-?3,SMAD2,SMAD3 expression at mRNA level,and Western blot at protein level.7.Tissue immunohistochemical and cell immunofluorescence staining was used to detect the expression of Notch 1 and TGF-?1 in keloid,hypertrophic scar and normal skin.The NFs,HSFs and KFs were treated with NC,condition control media and 12h-CM,24h-CM,and 48h-CM for 24 hours.Real-time PCR was used to detect Notch 1,Jagged-1(JAG-1),Jagged-2(JAG-2),Delta-1,TGF-?1 SMAD2,SMAD3 expression at mRNA level,and Western blot at protein level.Resultsl.The cell obtained from adipose tissue could express mesenchymal markers,and could also tansdiffermtiate into adipocytes and osteoblasts.2.Collected hADSCs conditioned medium did not cause cell apoptosis and cytotoxity.3.hADSCs conditioned medium inhibited the proliferation and migration of HSFs and KFs,and the proportion of cells in G0/G1 phase increased.4.hADSCs conditioned medium inhibited the fibrosis phenotype of HSFs and KFs.5.hADSCs conditioned medium inhibited the synthesis of extracellular matrix of HSFs and KFs,which inhibited the expression of type I collagen and fibronectin and secretion of hydroxyproline.6.After treatment of HSPs and KFs with hADSCs conditioned media,the expression of ACTA2 and protein decreased.7.After treatment of HSFs and KFs with hADSCs conditioned medium,the expression of Notchl/JAG-1 and TGF-?1/SMAD3 at mRNA and protein level were both significantly decreased in KFs,while only the expression of TGF-?1/SMAD3 at mRNA and protein level was significantly decreased in HSFs.Conclusion1.Cells cultured from adipose tissue were hADSCs,with potential multilineage differentiation abilities.2.hADSCs conditioned medium was safe to treat KFs and HSFs in vitro.3.hADSCs conditioned medium could inhibit the proliferation and migration of HSFs and KFs,but with no cytotoxicity and apoptosis.4.hADSCs conditioned medium inhibits the fibrotic phenotype of HSFs and KFs.5.hADSCs conditioned media could inhibit the synthesis of major extracellular matrix components of HSFs and KFs.6.After treatment of HSFs and KFs with hADSCs conditioned medium,myofibroblast transformation decreased.7.The treatment effect of hADSCs conditioned medium on the fibrotic phenotype of KFs may be related to the down-regulation of Notchl/JAG-1 and TGF-?1/SMAD3 signaling pathways,whereas in HSFs may be downregulation of TGF-?1/The SMAD3 signaling pathway play a role.