Proteomic Research On Schwann Cells

Part One Proteome of Schwann cellsObjective:Schwann cells(SCs)are the glial cells of the peripheral nervous system.They play a key role in peripheral nerve regeneration and are involved in multiple hereditary peripheral neuropathies.This study aimed to profile the proteome of primary cultured rat SCs using a high throughput proteomic methodology based on liquid chromatography coupled with mass spectrometry,which will provide a basis for a better understanding of SC biology.Methods:1、 Rat SCs was isolated from the sciatic nerve and purified by cytarabine and complement killing methods.2、 Total proteins of SCs were collected and protein concentration was determined by Bradford method.3、 The protein sample was precipitated by acetone and digested by trypsin.4、 Nano LC-MS/MS was performed and the raw data was processed by Protein Pilot software 3.0 to profile the SC proteome.5、 The main biological functions of identified proteins were annotated by Uniprot protein database,and the subcellular locations of identified proteins were assigned by IPA.6、 q RT-PCR was conducted to validate the expression of some proteins related to autophagy and other functions in SCs at gene level.7、 The expressions of CNP,GFAP,NGFR,TUBB3,NEFM and ATG5 were further confirmed by Western blot and immunocytochemical staining in primarily cultured SCs.Results:1、 The purity of the primary cultured SCs was over 95%.2、 A total of 1232 proteins were identified by mass spectrometry based proteomic strategy(FDR<0.1%).3、 These identified proteins could be categorized into multiple functional categories including transcription,signal transduction,neurogenesis and neural development,metabolism,immunity and inflammation,extracellular matrix,cell skeleton,chaperone protein,cell proliferation and migration,oxidative stress,apoptosis,axonal and synaptic proteins,cell cycle and cell split,transporters,cell adhesion,cell autophagy and SCs markers and other proteins.Subcellular analysis revealed that 49.7% of the proteins were localized in the cytoplasm,11.9% on the cell membrane,16.2% in the nucleus,4.6% in the extracellular,and 17.6% unknown.4、 The expression of Atg5 and other 21 proteins in SCs was verified by q RT-PCR.5、 The expression of CNP,GFAP,NGFR,TUBB 3,NEFM and ATG5 in SCs was confirmed by Western blot and immunocytochemical staining.Conclusions:1、 We set upped the proteomic methods for investigating SC proteome,and established the proteomic database of SCs.This study will not only contribute to our better understanding of SC biology,but also provide a database for further SC’s researches.2、 SCs were found to express proteins related to transcription,signal transduction,neurogenesis and neural development,metabolism,immunity and inflammation,extracellular matrix,cell skeleton,etc.3、 The neuronal markers NEFM,TUBB3 and autophagy related proteins were found to expressed in primary cultured rat SCs.Part Two Secretome of Schwann cellsObjective:The SC secretome was obtained based on the establishment of methods of investigating secreted proteins of primary cultured rat SCs.This study provides an experimental basis for further study of SC biological function.Methods:1.SCs were isolated from rat sciatic nerve and purified using cytarabine and complement killing methods.2.Typan staining was used to detect the cell viability of SCs cultured in serum-free medium whereby to optimize the time for harvesting SCs CM(conditioned Medium).3.CM of SCs cultured in serum-free medium for 8h was collected and purified and concentrated by ultrafiltration.The protein concentration of CM was determined by Bradford protein assay and the quality and integrity was estimated by SDS-PAGE electrophoresis.4.The protein sample of CM was precipitated by acetone and digested by trypsin.5.Nano LC-MS/MS was performed and the raw MS data was processed by Protein Pilot software 4.0.6.The biological function and subcellur location of these identified proteins were categorized according to Uni Prot knowledge database,GO and Pub Med.7.Expression of 39 proteins was validated by q RT-PCR at gene level.8.The expression of Cyr61,an angiogenesis associated proteins was confirmed in primary cultured SCs by immunocytochemical staining,and its involvement in the proliferation of SCs was determined by si RNA interference and CCK8.Results:1.615 proteins were identified by mass spectrometry(FDR<0.1%).2.According to the biological function of SCs,indentified proteins were fallen into the following categories: including cell death,necrosis,apoptosis,cell movement,morphology of cells,migration of cells,organization of cytoskeleton,cell survival,inflammation of organ,invasion of cells,development of vasculature,metastasis,angiogenesis,morphology of nervous system,neuritogenesis,growth of neurites,proliferation of neuronal cells,autophagy,axon guidance and axonogenesis,etc.Moreover,23.58% of the proteins were localized in extracellular exosome.21.30% in cytoplasm,15.77% in nucleus,14.31% in extracellular space,13.66% in cytosol and 9.27% in plasma membrane.3.The expression of the 39 indentified proteins including Cyr61 was verified by q RT-PCR.4.The expression of Cyr61 in the SCs was confirmed and the proliferation ability of SCs was found to be affected by suppressing the expression of Cyr61.Conclusions:1.The SC secretome was obtained based on the establishment of methods of investigating secretome of primary cultured rat SCs.2.SCs was found to secret proteins related to cell death,necrosis,apoptosis,cell movement,morphology of cells,migration of cells,organization of cytoskeleton,cell survival,inflammation of organ,etc.3.SCs were found to secrete proteins related to angiogenesis,cell invasion,and metastasis.Among these,SC proliferation was found to be affected by suppression of Cyr61.Part Three Comparison of the proteome of Sensory and Motor Schwann cellsObjective:Peripheral nervous system comprises sensory and motor nerves and their functions are different.It was evidenced that there are sensory and motor subtypes of SCs,but their difference in phenotype and function remained unclear.In this study,a quantitative proteomic analysis strategy based on isobaric tags for relative and absolute quantitation(i TRAQ)labeling was conducted to obtain an unbiased view on the proteomic difference of mouse sensory and motor SCs.This research will not only provide data basis to reveal the biological difference of sensory and motor SCs,but also will contribute to reveal the cellular and molecular mechanisms underlying the different regeneration properties of injured sensory and motor nerves.Methods:1.Breeding and genotyping of Tg(S100B-EGFP)1Wjt transgenic mice.2.According to anatomical structures,the sensory and motor nerves were dissected and digested then the obtained cells were cultured for three days.3.The sensory and motor SCs were sorted by flow cytometry.The proteins of sorted sensory and motor SCs were extracted.The protein concentration was determined by Bradford protein assay.The quality and integrity of protein samples were determined by SDS-PAGE.4.After overnight acetone precipitation,protein samples were digested,after purification,the obtained peptides were i TRAQ labeled and fractionated.5.Labeled samples were analyzed by nano LC-MS/MS,the raw data was processed by Protein Pilot software 4.0.6.The differentially expressed proteins were categorized into different biological functions and subcellur location according to the information provided by Uni Prot protein knowledge database,IPA and Pub Med.7.The gene expression of proteins differentially expressed in sensory and motor SCs was validated by q RT-PCR.8.A set of differentially expressed proteins in sensory and motor SCs were confirmed by Western blot and immunocytochemical staining.Results:1.Sensory and motor SCs of high purity were sorted by flow cytometry and proteins were extracted.2.A total of 6541 proteins were indentified(Unsused score≥1.3),among which,108 proteins were significant differentially expressed between sensory and motor SCs(Fold change≥2 or Fold change≤0.5),accounting for 1.65% of total identified proteins.In these differentially expressed proteins,85 were highly expressed in sensory SCs,while 23 were highly expressed in motor SC.3.The 85 proteins highly expressed in sensory SCs could be classified into the following functional categories,including protein metabolism,ubiquitin,chaperon,cell proliferation,cell cycle and cell division,energy metabolism,retinoic acid metabolism,cytoskeleton,transport,cell differentiation,development and growth,apoptosis,epigenetic repression,transcription,immune function,cell proliferation and adhesion,platelet-derived growth factor receptor,signaling pathway,binding,nerve growth and neurogensis and Others.While the other 23 highly expressed in motor SCs could be classified into cytoskeleton,neurogenesis,autophagy,binding,development,cell proliferation,cell migration,axon guidance,chromosomal stability,collagen catabolic process,axon guidance.4.The differential expressions of a set of proteins were validated by q PCR.5.In addition,higher expression of CNP,GAP43,and GMFB in sensory SCs,and higher expression of His1h4 a in motor SCs were validated by Western blot and immunocytochemical staining.Conclusions:1.Quantitative proteomic analysis revealed the differences between the proteome of sensory and motor SCs.2.A total of 108 differentially expressed proteins were identified,among which 85 were highly expressed in sensory SCs,while 23 were highly expressed in motor SCs.The main functions of 85 proteins were associated with protein synthesis,energy metabolism,transcription,growth and development,while those of of 23 proteins were involved in chromosome stability and extracellular matrix,etc.