Transcriptome Profile Of Rat Genes In Injured Spinal Cords After Adoptive Transfer Of M2 Macrophages By RNA-sequencing

Background:Our previous studies showed that spinal cord injury(SCI)can be detected early in the local microenvironment of nerve damaging M1 macrophages and nerve protective M2 macrophages,but a week after injury M1 cells accounts for absolute advantage,and M2 cell proportion is very few.The use of M2 cell adoptive immunity can improve the proportion of local M2 cells and play the role of neuroprotective effect.Its mechanism,however,is not yet clear.Study confirms that the SCI is heaviest inflammatory response time before and after a week,therefore,this study intends to 7 d after SCI as the point in time,analysis the M2 cells adoptive back to damage the influence of the local expression of gene transcription,as a new theoretical and experimental basis for clinical treatment of SCI.Objective:RNA-Seq was used to analyze the effects of m2-cells on the transcriptional expression of local genes after the SCI was 7D.Bioinformatics analysis,screening and identification of key molecular and signaling pathways.Method:(1)the separation of femoral bone marrow,adult SD rats by L929 conditioned medium produce the source of bone marrow macrophage cells(M0),M1 and M2 respectively by the conditions induce macrophages;(2)IHC was used to identify markers of M0,M1 and m2-type macrophages;(3)RNA-Seq analysis of the expression of M0,M1 and M2 macrophages at the transcription level;(4)q RT-PCR verification of RNA-Seq results;(5)using New York university weight injury instrument to prepare SD rat spinal cord injury model,and to be a macrophage immunized with m2 cell on that day;(6)the SD rats in the experimental group were executed 7 days after the SCI,and about 1cm of spinal cord tissues,including the injury center,were removed;(7)RNA-Seq was used to detect changes in the transcription level of local genes in SCI;(8)q RT-PCR verification of RNA-Seq results;(9)bioinformatics analysis,screening and identifying key molecules and signaling pathways.Results:(1)the macrophages cultured in L929 cell condition medium expressed CD68 and CD45,hardly expressing Arg1 and CCR7.CCR7 and CD68 were expressed in macrophages cultured in the medium of IFN-and LPS.The macrophages cultured in the culture medium containing il-4 and il-10 expressed M2 Arg1 and CD68;(2)The results of macrophage transcriptome analysis showed that M0 was compared with M1 group,and there were 2,535 differentially expressed genes in total,of which 1118 were up-regulated and 1,418 were down-regulated.M0 compared with M2,there were 2,229 differentially expressed genes,of which 694 were up-regulated and 1,436 were down-regulated.In total,there were 3107 differentially expressed genes between M1 and M2,of which 1336 were up-regulated and 1,772 were downregulated.GO enrichment analysis showed significant differentially expressed genes on immune system process,response to stress,response to external stimulus,inflammatory response,cellular response to chemical stimulus,cellular process,mitotic cell cycle,cellular nitrogen compound metabolic process,DNA metabolic process,etc.KEGG pathway analysis found that the most enriched pathways included ECM-receptor interaction,Antigen processing and presentation,TNF,p53,HIF-1,Fox O and NF-kappa B signaling pathways,Fructose and mannose metabolism,Galactose metabolism,Arginine and proline metabolism,etc.(4)the results of the analysis of the m2-type macrophage transcriptional group: compared with the sham operation group,there were 12 differentially expressed genes,including 5 upregulated and 7 down-regulated genes.Compared with the sham operation group,there were 4,304 different genes,2,321 of which were up-regulated and 1984 downregulated genes.In total,there were 2,986 differentially expressed genes,of which1,824 were up-regulated and 1163 were down-regulated,compared with the sham operation group.In total,there were 41 differentially expressed genes,31 of which were up-regulated and 10 down-regulated genes.GO enrichment analysis showed significant differentially expressed genes on nc RNA metabolic process,rRNA metabolic process,preribosome,CCR2 chemokine receptor bindin,etc.KEGG pathway analysis found that the most enriched pathways included TNF signaling pathway,Protein processing in endoplasmic reticulum,PI3K-Akt signaling pathway,NOD-like receptor signaling pathway,Chemokine signaling pathway,Antigen processing and presentation,etc.Conclusion:1.Successfully cultured and induced M0,M1 and M2 macrophages,and the purity of cells was consistent with subsequent experimental requirements;2.The RNA-Seq results of M0,M1 and M2 showed that M0 was compared with the M1 group,with a total of 2,535 differentially expressed genes,including 1118 upregulated and 1,418 down-regulated genes.M0 compared with M2,there were 2,229 differentially expressed genes,of which 694 were up-regulated and 1,436 were downregulated.In total,there were 3107 differentially expressed genes between M1 and M2,of which 1336 were up-regulated and 1,772 were down-regulated;ECM-receptor interaction,Antigen processing and presentation,TNF,p53,HIF-1,Fox O and NFkappa B signaling pathways,Fructose and mannose metabolism,Galactose metabolism,Arginine and proline metabolism,etc is the main enrichment pathway.3.After intravenous injection of the immune SCI rats,the M2 cells can infiltrate into the injured spinal cord.4.The results of RNA-Seq of m2-macrophage adoptive immune system showed that there were 41 differentially expressed genes in the SCI injury group compared with the m2 macrophage relay group,with 31 up-regulated and 10 down-regulated genes.TNF,endoplasmic reticulum protein processing,PI3K-Akt,NOD-like receptor,antigen processing and presentation are typical enrichment signaling pathways.