ITRAQ-based Proteomics Of Bone Marrow Mesenchymal Stem Cells After Co-cultured With Schwann Cells In Vitro

Objective:Spinal cord injury(SCI)is a traumatic disease of the central nervous system,accompanied with high incidence and high mordidity.Presently,there is no effective treatment for it.The research team have found that the activated Schwann cells(SCs)could secrete neurotrophic factors and inhibit neuronal apoptosis after SCI,Besides,SCs could induce bone marrow mesenchymal stem cells(BMSCs)to differentiate into neurons after co-transplantation in vivo and then promote axonal regeneration and functional recovery of patients with SCI.With the application of iTRAQ(isobaric Tag for Relative and Absolute Quantitation),we could deeply find that protein changes of BMSCs after co-culture with SCs,and then establish the differential protein profiles.Based on that,we can look for potential cellular signal transduction pathways involved in repair of SCI,establish the key regulatory sites in the cell signaling pathways,and further clarify the mechanism of co-transplantation of BMSCs and SCs,Methods:The tissue mechanical separation and trypsin digestion method were used for culture of Schwann cells,the rapid trypsin digestion method and double 30 minutes differential adherence method were used for purification of SCs.BMSCs were isolated and cultured by modified whole bone marrow adherence method.SCs were identified by S100 immunofluorescence staining,and BMSCs were identified by flow cytometry and three cell line differentiation.3 groups were established in the experiment: single BMSCs group(SCs,0d),co-culture of BMSCs and SCs for 3 days(SCs,3d),co-culture of BMSCs and SCs for 7 days(SCs,7d).The method of semi quantitative medium exchange was applied for co-culture.Protein was extracted by the method of lysis liquid extraction.The protein concentration was determined by 2D Quant kit.The differential proteins were identified by iTRAQ/TMT mass spectrometry.The qualitative and quantitative calculation of the two level spectrum informations were determained with MSCot software.Annotation and functional analysis of GO,KEGG and Interpro term functions was made separately for the differential proteins.Additional,significant enrichment analysis was made for the differential proteins.Network interaction analysis of the differential proteins was determained with application of STRING database.Results:In the study,differentially expressed proteins were dedined as those expressed with up-regulated or down regulated more than 1.3 times.In SC3 d vs SC0 d group,29 proteins were up-regulated and 45 proteins were down-regulated.In SC7 d vs SC0 d group,43 proteins were up-regulated and 32 proteins were down-regulated.In SC7 d vs SC3 d group,83 proteins were up-regulated and 24 proteins were down-regulated.The GO analysis revealed that differentially expressed proteins involved 13 kinds of biological processes,9 kinds of cell components,and 9 molecular functions.The subcellular localization analysis revealed that differentially expressed proteins mainly located in the nucleus,cytoplasm,extracellular protein,plasma membrane.The enrichment analysis revealed that functional terms with significant enrichment were relatively less in SC3 d vs SC0 d group.Regarding biological processes,differentially expressed proteins mainly enriched in lipid metabolism,carbohydrate metabolism,biodegradation in SC7 d vs SC0 d group.Meanwhile,regarding cell components those mainly enriched in lysosome,cytoskeleton and extracellular matrix.Those mainly enriched in hydrolase activity,glycosaminoglycan binding and carbohydrate binding for molecular functions.Significantly enriched KEEG pathways in three groups were mainly glycosphingolipid biosynthesis,sphingolipid metabolism and lysosomal metabolism.Significantly enriched protein domains were glycoside hydrolase catalytic domain,calmodulin homology domain and glycosyl hydrolase family 13.The enrichment cluster analysis revealed that 54 biological processes terms,38 cell components terms and 38 molecular functions terms could be clustered respectively.KEEG cluster analysis mainly concentrated in SC7 d vs SC3 d group,and the relevant pathways were mainly sphingomyelin biosynthesis-ganglion,lysosomal metabolism and sphingomyelin metabolism.The interpro cluster analysis mainly concentrated in SC7 d vs SC3 d group,and the relevant domains were mainly glycoside hydrolase superfamily,sulfatase and glycoside hydrolase catalytic domain.Conclusions:With quantitative proteomics technology in the research,differential protein expression profiles of BMSCs was drawn after co-cultured with SCs.This lays a foundation for revealing the deep repair mechanism after co-transplation of BMSCs and SCs to the SCI site,for the study of these problems will help to deepen and expand the clinical application of stem cells.