Isolation,Identification And Proteome Study Of Exosomes Derived From Human Olfactory Mucosa Mesenchymal Stem Cell

Human Olfactory Mucosa Mesenchymal Stem Cells(OM-MSCs)are ideal seed cells for clinical treatment.The paracrine effect of OM-MSCs is explained as the main mechanism for tissue and nerve repair function.Exosomes as an important extracellular vesicle secreted by OM-MSCs,have important clinical value in studying their biological characteristics.Exosomes are a kind of nano-sized membranous vesicles,which carry various proteins derived from mother cells.Although functional molecules such as nucleic acids and lipids contained in exosomes are studied hot,protein is the most direct and efficient embodiment of life function.The role of exosomal proteins in exosome-mediated signaling and signal regulation can not be ignored.Therefore,in this study,we first isolated and cultured OM-MSCs from human olfactory mucosa by tissue adherence method.The exosomes in the culture supernatant of OM-MSCs were isolated and purified by ultracentrifugation.The surface markers,particle concentration,particle size distribution,and morphological characteristics of the exosomes were analyzed and characterized.The results showed that the isolated exosomes of OM-MSCs were consistent with the characteristics of general exosomes,the purity was high and suitable for further proteome analysis.In order to explore the protein composition and potential functions of OM-MSCs-derived exosomes,this study performed proteome analysis by LC-MS/MS mass spectrometry and verified the functional experiments.A total of 304 OM-MSCs-derived exosomes were identified by mass spectrometry.The subcellular localization of these proteins is mainly extracellular vesicles and exosomes,accounting for 80.4% of the total protein.which acted mainly on the cell membrane to bind receptors and can participate in the positive regulation of biological processes and phylogenetic processes.In the constructed interaction network of candidate proteins,a large number of potential candidate proteins related to cell proliferation,apoptosis,angiogenesis,neurotrophicity and immune regulation were screened.By comparing the secretome data of OM-MSCs,the core role of OM-MCSs-derived exosomes in the paracrine treatment mechanism was revealed from the perspective of proteome.These candidate proteins are involved in the signaling pathway of tissue wound repair,the main physiological phenomenon of tissue wound repair is accompanied by angiogenesis due to proliferation of endothelial cells.Related functional experiments show that OM-MSCs-derived exosomes can be taken up by Human Brain Microvascular Endothelial Cells(HBMECs)and further promote the HBMECs proliferation,migration and angiogenesis in vitro.Combining with proteome data for mechanismanalysis,it may be mainly caused by the phosphorylation of key protein kinase AKT by HSP90,EGFR,PDGFRB and other molecules in the exosomes of OM-MSCs,which activates the PI3K/AKT signaling pathway,This suggests that OM-MSCs derived exosomes have the potential to treat ischemic brain damage caused by stroke.The experimental data of this study also provides an important reference for the clinical treatment based on OM-MSCs transplantation.