Study On Proteomics Analysis Of Schwann Cells In Nerve Injury

[Objective]Schwann cells(SCs)are myelin-forming cells in the peripheral nervous system and play crucial roles in peripheral nerve regeneration.SCs are transformed into activated Schwann cells(ASCs)after peripheral nerve injury,secreting various growth factors to promote axon growth and neuronal survival,phagocytizing myelin sheath and forming axonal regeneration tracks.Although SCs have also been widely used as candidate cells in cell transplantation repairment,it's specific mechanism still remains unclear.To elucidate the mechanisms of peripheral nerve injury repair,this study carried out proteomic analysis of SCs before and after peripheral nerve injury.[Methods]Unilateral sciatic nerves were ligated in adult Wistar rats.Harvesting the primary ASCs from the ligating side of sciatic nerves,while the normal Schwann cells(NSCs)from the contralateral sciatic nerves seven days later.Passaging the cells to the third generation using purification passaging method by collagenase II.Identifying the purity of obtained SCs by immunofluorescence staining with S-100.Extracting the proteins of ASCs and NSCs respectively,performing proteomics analysis using tandem mass spectrometry combined with liquid chromatography-tandem mass spectrometry technology.Eventually screening the significant differentially expressed proteins with the fold change greater than 1.5 times and the P value less than 0.05.Performing Gene Ontology analysis,Kyoto Encyclopedia of Genes and Genomes analysis,protein-protein interaction analysis by inputting significant differentially expressed proteins into Blast2 GoPro software.Selecting several most significant differentially expressed proteins,performing Western Blot experiments to analyze the protein expression and RT-PCR experiments to analyze the relative RNA expression in two groups,to verify the correctness of the results.[Results]1.Schwann cells purification.After 5-7 days of primary culture,the primary SCs confluence degree was over 80%.Collagenase II purification passaging method can effectively purify SCs,and there is no negative staining in two type of cells when the third generation SCs were confirmed by S-100 immunofluorescence staining2.Proteomics analysis.In this study we obtained 31477 unique peptides,and 5574 proteins,including 4473 quantifiable proteins;and 122 differentially expressed proteins were identified,including 72 up-regulated proteins and 50 down-regulated proteins using TMT combined with LC-MS/MS proteomic technology after the extraction of ASCs and NSCs proteins.The KEGG analysis results shown that differentially expressed proteins were mainly enriched in Purine metabolism,Biosynthesis of antibiotics,Amino sugar and nucleotide sugar metabolism,and the GO analysis results show that the difference expressed proteins were enriched in biological process of cell differentiation,cellular nitrogen compound metabolic process,response to stress,and enriched in cell component of plasma membrane,cytosol,extracellular space,and enriched in molecular function of ion binding,enzyme binding,enzyme regulator activity.The GO-enzyme analysis showed that the differentially expressed proteins contained a large number of Oxidoreductases and hydrolases.3.Experimental result verification.The three proteins of Alb,Spp1 and Pten were selected for Western blot analysis.The results showed that the relative gray values of Alb and Pten of ASCs were higher than that of NSCs.The relative gray value of Spp1 was lower than that of NSCs.The P values were 0.0018,0.0006 and 0.0363 respectively.RT-PCR analysis of four genes including PTEN,SPP1,COMP and ALB showed that ASCs compared with NSCs,the relative expression ratio of PTEN,COMP and ALB was greater than 1,while SPP1 less than1.The P values were 0.0070,0.0118,0.0004,0.0063 respectively.According to the level of P <0.05,the difference was statistically significant.The above results were consistent with proteomics information.[Conclusions]1.Collagenase II can effectively purify the SCs,improve purification efficiency by differential detachment method.2.ASCs and NSCs have significant differences in protein expression level.The differentially expressed proteins are enriched in the pathway of Purine metabolism,Biosynthesis of antibiotics,Amino sugar and nucleotide sugar metabolism,which suggest that nerve repair is associated with these biological processes.And the process of cell differentiation and nitride metabolism may play key roles in process of nerve repair.The peripheral nerve repair process includes activation of a large number of enzymes,of which oxidoreductases and hydrolases are mostly.