| Background and objective:Autologous fat grafting is widely used for various treatments in plastic surgery,due to its good biocompatibility,low tissue rejection rate and allergic reaction rate.However,the unstable retention of fat grafts has been a key clinical problem to be overcome.Adipose tissue has poor tolerance to ischemia,and the transplanted adipose tissue needs to rebuild blood supply at an early stage in order to survive stably.Therefore,enhancing hematologic reconstruction in the early stages of fat grafting is one of the key factors to promote graft survival.Although stem cell therapy has high application value currently in promoting vascularization,its risk of promoting tumor formation and difficulties in transformation technology considerably limit the clinical application of stem cells.Therefore,finding key active ingredients that can replace stem cells directly and have vasogenic effects can avoid the above potential problems to the greatest extent.Exosome(Exo)is a class of membranous vesicles about 30-150 nm in diameter released into the extracellular matrix from cells.It can carry a variety of biological components,such as proteins,lipids and non-coding RNAs,and regulate the biological activity of target cells through membrane fusion or endocytosis.Numerous studies have applied human adiposederived stem cells exosome(h ADSC-Exo)to ischemic injury diseases,showing a good angiogenic effect similar to human adipose stem cells(h ADSCs).What’s more,h ADSCExo has the advantages of low immunogenicity,no risk of tumor formation,high stability and easy storage and transportation.Our early study found that there were some differentially expressed mi RNAs(DEmi RNAs)between h ADSC-Exo and human foreskin fibroblast exosome(HFF-Exo).These DE-mi RNAs may significantly promote the angiogenesis of artificial dermal prefabricated skin flap by regulating the function of some genes.Therefore,whether h ADSC-Exo can improve the retention ability of fat grafts through these DE-mi RNAs and its potential regeneration mechanism have aroused our strong interest.In this study,h ADSC-Exo and HFF-Exo were co-cultured with human umbilical vein endothelial cells(HUVEC)or h ADSCs in vitro to investigate the effects of two kinds of exosome on the function of HUVEC and adipogenic differentiation of h ADSCs.By establishing a nude mouse transplantation model and co-transplanting these exosomes and fat grafts,this study explored the promoting effect of h ADSC-Exo on the survival of fat grafts and its potential mechanism.Methods:1.The adipose tissue obtained by liposuction was cut into pieces and placed in the prepared digestive extract.After digestion,grinding,sieving,centrifugation and other steps,the cells suspended in the complete culture medium and were cultured on the plate.The morphology of primary h ADSCs was observed under light microscope,the specific markers on the surface of h ADSCs were identified by flow cytometry,and the osteogenic and adipogenic differentiations of h ADSCs were induced to identify the potential of multilineage differentiation.2.The primary h ADSCs and Human foreskin fibroblasts(HFFs)were cultured in complete medium without Exo.The supernatants from the third to the sixth generations of h ADSCs and HFFs were collected respectively,and then the Exo in the supernatants was isolated and purified by ultracentrifugation.The morphological characteristics of the Exo were observed by transmission electron microscope,the sizes of Exo were detected by nanoparticle tracking analysis,and the expressions of Exo membrane proteins(CD63 and CD81)were detected by Western blotting.3.The nuclei of HUVEC were labeled with DAPI and the labeled HUVEC cells were co-incubated with h ADSC-Exo and HFF-Exo labeled with PKH67 green fluorescent dye.The uptake of Exo by HUVEC cells was observed under inverted fluorescence microscope.4.The co-culture system of h ADSC-Exo and HFF-Exo with HUVEC and h ADSCs was established.First of all,the effects of different concentrations of h ADSC-Exo on the proliferation of HUVEC were detected by CCK-8 cell proliferation assay,and the appropriate concentration of Exo was determined for subsequent experiments in vitro and in vivo.Then the effects of two exosomes on the proliferation and migration of HUVEC were evaluated by Edu cell proliferation assay and Transwell cell migration assay,and the adipogenic differentiation induction experiment was used to evaluate the effects of two exosomes on the adipogenic differentiation of h ADSCs.5.The nude mice model of fat transplantation was established and divided into h ADSCExo group and HFF-Exo group.The fat grafts of the two groups were intervened differently.Then,the paraffin sections of fat grafts were used for HE,immunohistochemical staining and immunofluorescence staining to evaluate the retention and neovascularization of fat grafts,and adipogenic differentiation of h ADSCs in fat grafts at the tissue and cellular level.Finally,with the help of bioinformatics methods,the potential molecular mechanism of fat graft survival was explored,and the expression of VEGFA protein and the activation of Wnt/β-catenin signal pathway were detected by Western blotting.Results:1.The primary extracted h ADSCs divided and proliferated and showed long fusiform.After subculture,the cell morphology was uniform and grew in spirals and clusters.Flow identification of h ADSCs’ surface specific markers showed that CD29,CD44 and CD90 were positive,and the expression rates were 99.10%,98.61% and 99.04%,respectively.The positive expression rates of CD34 and CD45 were 0.74% and 0.49%,respectively.The multilineage differentiation experiment of h ADSCs showed that the extracted h ADSCs was successfully induced to differentiate into adipocytes and osteoblasts with the characteristics of multidirectional differentiation.2.The morphology of h ADSC-Exo and HFF-Exo under transmission electron microscope showed that these exosomes were round or oval with phospholipid bilayer structure.The results of nano-particle tracking analysis showed that the average particle size of h ADSC-Exo was 139.5 ± 93.3 nm and of HFF-Exo was 131.0 ± 52.2nm.Western blotting showed that the Exo expressed CD63 and CD81.The above results prove that Exo has been successfully isolated and can be used for the follow-up researches.3.The experiment of co-incubation of fluorescence-labeled Exo with HUVEC under inverted fluorescence microscope showed that PKH67-labeled Exo could be seen in the cytoplasm of DAPI-labeled HUVEC,indicating that h ADSC-Exo and HFF-Exo could be absorbed by HUVEC.4.The result of CCK-8 proliferation experiment showed that the medium of 100 μg/ml h ADSC-Exo promoted the proliferation of HUVEC most obviously.Through Ed U proliferation assay and Transwell migration assay,it was found that compared with HFFExo,h ADSC-Exo could promote the proliferation and migration of HUVEC cells more effectively(P<0.01).The adipogenic differentiation induction experiment showed that compared with HFF-Exo,h ADSCs-Exo could maintain and promote the adipogenic differentiation ability of h ADSCs more efficiently.5.The result of fat transplantation model in nude mice showed that the fat grafts treated with h ADSC-Exo were superior to HFF-Exo in weight,volume,fat density and morphological and structural integrity of fat.The results of immunofluorescence and immunohistochemical staining of graft paraffin sections showed that the intensity and proportion of PPARγ fluorescence expression,the number of CD31 positive vessels and Ki67 positive proliferating cells in adipose grafts treated with h ADSC-Exo were significantly increased(P<0.05).6.Through bioinformatics methods,a total of 605 key genes(TGs)that may be targeted and regulated by h ADSC-Exo in the process of adipogenic differentiation of h ADSCs were identified.Through GO annotation and KEGG pathway enrichment analysis,it was found that these TGs were directly related to the biological process of extracellular matrix.Through the PPI network,it was found that CTNNB1,a key regulator of Wnt signal pathway,was the gene with the highest node degree in the network.Western blotting showed that compared with HFF-Exo,the expression of VEGFA protein was up-regulated while the expression of Wnt/β-catenin pathway was down-regulated in h ADSC-Exo treated group(P<0.01).Conclusion:Our results have shown that h ADSC-Exo can enhance the angiogenesis of adipose grafts,and maintain and raise the adipogenic differentiation ability of h ADSCs in the grafts by inhibiting the Wnt/ β-catenin pathway,and jointly promote the retention of adipose grafts.This finding provides a theoretical basis for the clinical transformation of h ADSC-Exo in fat transplantation and a new idea for further understanding of the molecular mechanism of h ADSC-Exo in fat graft survival. |
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