| Objectives:This study is aimed to filter out differential expressed proteins in injured tissue microenvironment at different time points after transplantation of bone mesenchymal stem cells(BMSCs)combined with Activated Schwann cells(ASCs)for spinal cord injury(SCI)through iTRAQ proteomics technique,to make functional annotation of these proteins,to predict the involved biological processes and potential signaling pathways related to nervous regeneration,to initially explore the mechanism of cell transplantation for SCI at molecular level,to uncover key regulatory proteins and their roles in this process,and finally to provide novel ideas and theoretical basis for optimizing the cell transplantation strategy and clinical transforming application.Methods:1.Primary cell culture: BMSCs were directly isolated and cultured from bone marrow of Wistar rats identified by flow cytometry.After pre-injury of sciatic nerve,ASCs were dissociated and cultured from tissue block by digestion of collagenase,and were identified by immunofluorescence staining of S100.2.Experimental grouping: 54 adult female Wistar rats were randomly divided into 6 groups: A.control group treated with DMED(7d);B.control group treated with DMED(14d);C.control group treated with DMED(28d);D.cell transplanted group with BMSCs and SCs(7d);E.cell transplanted group with BMSCs and SCs(14d);F.cell transplanted group with BMSCs and SCs(28d).3.Establishment of SCI animal model and cell transplantation: the use of NYU Impactor II was used to establish T10 spinal cord injury model of Wistar rats with the weight of 10 grams and height of 2.5cm.DMEM,ASCs mixed with BMSCs were respectively contained in 10 μl Hamilton microinjector,and injected into the area of spinal cord injury at 7 d post injury with dose of 15 μl and cell density of 1×105 / μl.Then rats were respectively sacrificed at 7 d,14 d,28 d post transplantation.After heart perfusion,a 0.5cm segment of spinal cord around injured site were dissected.4.Proteomics analysis: After extraction of protein by lysate,2D Quant kit was used to determine the concentration of protein in each group.iTRAQ/TMT mass spectrometry were used to quantify differential expressed proteins.The qualitative and quantitative calculation on the secondary mass spectrum information was carried out by Mascot software.The functional annotation and clustering of differential expressed proteins were performed through GO analysis,and the network interactions between these proteins were analyzed by STRING database.Results:At 7 d after transplantation of BMSCs and ASCs for SCI,the total number of differentially expressed proteins was 105,among which 39 were up-regulated and 66 were down-regulated.The number of differentially expressed proteins at 14 d was 185,among which 151 were up-regulated,34 were down-regulated;the number of differentially expressed proteins at 28 d was 81,among which 46 were up-regulated and 35 were down-regulatedThe GO annotation and cluster analysis showed that differential expressed protein at 7d after transplantation were mainly enriched in inflammatory reaction,immune response,acute phase reaction and other immune-related biological processes;differential expressed protein at 14 d after transplantation were mainly enriched in the biological processes such as synaptic and neurotransmitter transport;differential expressed protein at 14 d after transplantation were mainly enriched in extracellular space remodeling,extracellular matrix,laminin,cell adhesion,myelination and other biological processes and cell components.String network interaction analysis showed that the down-regulated STAT1 protein was located at the core position of the protein interaction network at 7 days after transplantation.Conclusions:In this study,the dynamic protein expression profile in microenvironment of injured spinal cord after transplantation of BMSCs and ASCs was initially portrayed.This study uncovered regenerative promotion of BMSCs and ASCs transplantation for SCI were mainly mediated by inhibition of inflammatory and immune response,antiapoptosis,modulation of microenvironment,and indicated several key proteins,such as STAT1 may play an important role in cell mediated spinal cord regeneration through immune regulation and anti-apoptosis.These results suggested that transplantation of BMSCs and ASCs may play an important role in modulating the injured microenvironment of spinal cord.Deep elucidating the molecular mechanism of BMSCs and ASCs transplantation is beneficial for optimizing the transplanting strategy and building the theoretical foundation of clinical application of cell transplantation for repairing spinal cord injury. |
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