| ?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 Microarray technique,to make functional annotation of these genes,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 S 100.2.Experimental grouping:10 female Wistar rats of 4 weeks at the was used to extract bone marrow mesenchymal stem cells and schwann cells,which weight was between(80±10)g and the two cells used for transplantation were from the same rats.54 adult female Wistar rats at the SPF level 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 Model 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 a Hamilton microinjector,and injected into the area of spinal cord injury at 7 d post injury with dose of 15?l and cell density of 1×10~5?lThen 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:At the corresponding time,the RNA was extracted using Trizol from the spinal cord of rats.Using the extracted RNA synthesized cDNA,and Using cDNA synthesized and augmted cRNA.After purification?scanning and chip hybridization of cRNA,we got the final data.According to the standard that foldchange multiples greater than 2 and p is less than 0.05,we got differences related genes.The difference genes were analyzed by GO function enrichment and KEGG pathway.?Result?In the 7 days after BMSCs combined with ASCs cells,there were 44differentially expressed genes,of which 20 were up-regulated and 24 were down-regulated.There were 606 difference genes in the 14 days after transplantation,and 386 were up-regulated,and 218 were down-regulated.The number of differentially expressed genes in the 28-day group was 50,with 23 and 27respectively.?Conclusion?1.Through bioinformatics analysis,some key genes related to spinal cord injury repair were screened.Among them,Ppp1r12a,Npc1 and Ngb gene reached significant difference,and inhibit apoptosis,improve local microenvironment,Nrsn1,Cyp26a1 Mmp12 genes related to promote axonal regeneration and promote synaptic plasticity,S100A8/A9 genes regulated cell proliferation and apoptosis,inflammation in the body.2.In 7 days after in seven days after transplantation,cells inhibit the inflammatory reaction,resistance to apoptosis,in 14 days after transplantation,cells promote axon regeneration,synaptic plasticity,in 28 days after transplantation,there appears the regeneration of new protein,cell replication,etc... |
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