P75NTR Overexpressed Epidermal Stem Cell Conditioned Medium Promotes Wound Healing By Inhibiting The Expression Of Sortilin In Fibroblasts

BackgroundsThe skin defects caused by burn and trauma can bring about local pain,infection and the subsequent pathological scar even shock and death.How to accelerate the epithelialization,close the wound as early as possible is a main goal of wound healing treatment.Epidermal stem cells(ESCs)and fibroblast(Fb)both are important cells in wound healing.ESCs can not only maintain daily metabolism of the skin but also generate new epidermal cells after trauma.However,clinical application of stem cells may be limited for the reason of poor survival after administration and risk of inducing tumor.Alternative therapeutic strategies with stem cells conditioned-medium containing many stem cell paracrine factors are required to solve the previous problems.After trauma,Fb as another important repairing cell began to migrate from the edge to the center of the wound and combined with neovascularization and inflammatory cells to form a granulation tissue to fill the wound.But excessive proliferation of Fb in the shaping stage can lead to the formation of pathological scars.The nerve growth factor receptor P75NTR as the first discovered neurotrophin receptor is a low-affinity receptor of nerve growth factor(NGF).It eventually leads to cell survival or apoptosis depending on its co-receptor,adaptor protein and signaling pathway.The co-receptors have been confirmed as Trk,Nogo and Sortilin.Although the main function of P75NTR is to regulate cell survival,death and intercellular signaling in neuronal system,the role of P75NTR in non-neurological system has also received increasing attention.In recent years,P75NTR has been more deeply researched as a surface marker,which also suggested a close relationship between P75NTR and tissue repair.Our previous experiments have confirmed that P75NTR promoted the proliferation,migration and differentiation of ESCs in vitro and accelerate wound healing in vivo.Whether P75NTR can influnce the secretory function of ESCs is not deeply researched.For there was no significant difference in P75NTR between P75NTR low expression vector and P75NTR normal expression vector,so we selected P75NTR overexpressed ESCs conditioned medium(ESC-CM)to observe the effect of P75NTR on ESCs secretory function.Sortilin as a co-receptor of P75NTR plays an important role in cell apoptosis.The most obvious feature of pathological scar is hyperfibrosis resulted from less apoptosis of Fb.Our previous study has shown that the expression of Sortilin in human keloid fibrobalsts(KFs)were lower than that of normal skin fibroblasts(NFs).It is further proved that overexpression of Sortilin can accelerate the apoptosis of KFs.These suggest that low expression of Sortilin in keloid may result in decreased apoptosis of Fb and excessive deposition of collagen.Therefore,we hope to observe the effect of P75NTR on secretory function by obtaining ESC-CM.Furthermore,we hope to explore whether P75NTR overexpressed ESC-CM has an effect on wound healing through affecting Sortilin of cultured mice wound Fb by its containing secretory growth factors.ObjectiveTo analyze the relationship among P75NTR,ESC-CM,mice wound Fb and Sortilin in order to investigate the effect of P75NTR on the secretory function of ESCs in vitro.And we hope to explore the new wound healing intervention method by observeing the effect of P75NTR overexpressed ESC-CM on mice wound in vivo and on Sortilin of mice wound Fb cultured in vitro.Methods1.Obtainmen of mice P75NTR overexpressed ESC-CM1.1 Isolation and culture of mice ESCsMice ESCs were isolated and cultured by trypsin-EDTA digestion and type ?collagen rapid adhension method.Morphology of cells were observed by inverted phase contrast microscope and surface markers such as Integrinp1,CK19,P75NTR and CK10 were detected by flow cytometry to identify ESCs1.2 Lentivirus tansfection of mice ESCsESCs of passage 2(1 X 104)were transfected with P75NTR carrying lentivirus and empty lentivirus constructed by genetic engineering method.Next they were divided into experimental group and negative control group respectively.Immunofluorescence assay was employed to detect the efficiency of transfection with green fluorescence.The number of P75NTR expression in ESCs was further estimated by Immunocytochemistry and Western Blot.1.3 Secretory function of mice ESCsELISA method was used to estimate the secretory function of ESCs by measuring TGF?1,VEGF and EGF.2.Effect of P75NTR overexpressed ESC-CM on mice wound Fb2.1 Formation of full-thickness skin defect model in miceC57BL/6 mice(6 weeks old,male,20-25g)were anesthetized,shaved and wiped.Two full-thickness skin defects were made with a 1cm diameter biopsy punching on back of each mouse.2.2 Grouping and treatment of mice woundsAll mice were randomly divided into 3 groups(n=6),an equal amount of P75NTR overexpressed ESC-CM,P75NTR normal-expressing ESC-CM and empty medium without ESCs were respectively injected around the wound at Od and 3d after injury.They were served as experimental group(P75NTRvoESC-CM),negative control group(Control)and blank control group(Vehicle)respectively.2.3 Assessment of mice woundsThe area of wound was recorded on the 0d,3d and 7d after injury by transparent film tracing method and the rate of unhealed wound was calculated.On the 7d after injury,the skin including 2mm wound edge was observed by HE staining for histological changes,Masson staining was employed to observed the morphology and number of collagen.3.Effect of P75NTR overexpressed ESC-CM on the expression of Sortilin in mice wound Fb cultured in vitro3.1 Isolation and culture of mice wound FbMice Fb was obtained from wound center 7 days after injury,isolated by trypsin-EDTA digestion and cultured respectively with DMEM,DMEM+Vehicle,DMEM+Control,DMEM+P75NTRvoESC-CM.The morphology of the cell was observed by inverted phase contrast microscopy and the markers such as Vimentin and Keratin were detected by flow cytometry to identify Fb.3.2 Detection of expression of Sortilin of mice wound Fb in different mediaWestern Blot was used to estimate the expression of Sortilin in mice wound Fb cultured in four different conditioned media.3.3 Survival assessment of mice wound FbCell Counting Kit-8(CCK-8)was employed to estimate the survival of mice wound Fb in four different conditioned media.4.Statistical analysisThe above data expressed as mean ±standard deviation were statistically analyzed with SPSS 17.0 statistical software.Data comparison between two groups was performed with Student's t-test and data among multiple groups was performed with one-way analysis of variance(ANOVA).*P<0.05 and**P<0.01were both considered as statistically significant difference.Results1.Obtainment of mice P75NTR overexpressed ESC-CM1.1 Isolation and culture of mice ESCsESCs isolated and cultured with above methods had strong cloning ability and presented as typical paving stone.Specific surface markers of ESCs including Integrin?1,CK19 and P75NTR were highly expressed while CK10 as the surface of keratinocyte has a low expression.1.2 Lentivirus tansfection of mice ESCsESCs were transfected with P75NTR carrying lentivirus and empty lentivirus respectively.The green fluorescence was detected to identify the successful transfection.Furthermore,higher levels of P75NTR was detected in ESCs transfected with P75NTR carrying lentivirus by Immunocytochemistry and Western Blot,indicating P75NTR overexpressed lentivirus vector construction was successful and can effectively tansfect ESCs(*P<0.05).1.3 Secretory function of mice ESCsIt was found that the number of TGF?1 and VEGF in P75NTR overexpressed ESCs was significantly higher than that in control group(**P<0.01)while EGF is on the contrary(*P<0.05).2.Effect of P75NTR overexpressed ESC-CM on mice wounds2.1 General view of mice woundsThe wounds were observed at 0d,3d and 7d after injury.It was seen that there was no significant difference among three groups all with a small amount of exudation at Od after injury.At 3d after injury,all wounds of three groups showed obvious contraction.The P75NTRvoESC-CM group was dry and has the most contraction,followed by the Control group.The Vehicle group had the lightest contraction and the moist wound surface.At 7d after injury,wounds of P75NTRvoESC-CM group recovered substantially while the Vehicle group had the largest residual wounds.The rate of unhealed wound in P75NTRvoESC-CM group was the lowest among three groups which were calculated at Od,3d and 7d after injury(*P<0.05).The rate of unhealed wound in Control group was significantly lower than the Vehicle group(*P<0.05).2.2 HE stainingAt 7d after injury,the thickest epidermal layer of wound edge and the most migratory cells were detected in P75NTRvoESC-CM group.The migratory cells lying in the basal layer were deeply stained in cytoplasm and nucleus and presented a proliferating state of increased volume and loose arrangement.The changes of Control group were stronger than the Vehicle group(**P<0.01)but weaker than the P75NTRvoESC-CM group(**P<0.01).2.3 Masson staining of collagenThe collagen deposition was observed with Masson staining at 7d after injury.The collagen in the granulation of the P75NTRvoESC-CM group was thick,deeply stained and closely arranged.And the positive area ration of collagen was significantly higher than two other groups(**P<0.01).The Vehicle group has the least collagen which was thin,slightly stained and sparsely arranged.The change of Control group was in the middle.3.Effect of P75NTR overexpressed ESC-CM on the expression of Sortilin in micewound Fb cultured in vitro3.1 Isolation and culture of mice wound FbMice wound Fb isolated and cultured has strong cloning ability.It presented the morphology of typical shuttle.Green fluorescence of Vimentin and empty fluorescence of Keratin proved successful isolation and culture of mice wound Fb.3.2 Detection of expression of Sortilin of mice wound Fb in different mediaIt was found that sortinlin of mice wound Fb in DMEM and DMEM+Vehicle group were higher than other groups while the difference between themselves was not statistical(P>0.05).The Sortilin of mice wound Fb in DMEM+Control was significantly lower than above two groups(*P<0.05)and that in DMEM+P75NTRvoESC-CM group was the lowest(*P<0.05).3.3 Detection of survival of mice wound Fb with CCK-8 in different mediaThe results showed that there was the most surviving Fb in DMEM+P75NTRvoESC-CM group(*P<0.05)followed by DMEM+Control group.The surviving Fb in DMEM+Vehicle group and DMEM group were less than above two groups with no significant difference between themselves(P>0.05).Conclusions1.P75NTR is positively expressed in mice ESCs cultured in vitro.ESCs with overexpression of P75NTR can secret more TGF?1and VEGF but less EGF.2.ESC-CM with P75NTR overexpression can improve the wound healing of full-thickness skin defect.The thickness of epidermis and collagen deposition were promoted at 7d after injury.These indicate that P75NTR overexpressed ESC-CM can promote fibroblasts to secrete more collagen to fill the wound bed and accelerate the epithelization through the growth factors secreted by ESCs.3.P75NTR overexpressed ESC-CM can inhibit the expression of Sortilin in mice wound Fb and promote the survival of mice wound Fb,which may attribute to the growth factors secreted by P75NTR overexpressed ESCs.