Extraction And Identification Of Serum Exosomes And Analysis Of MiRNA Expression Profiles In Severe Burns Rats During Shock Stage

BackgroundSevere burns are a fatal blow to the body.After severe burns,a large amount of tissue necrosis,obvious stress,shock,nutritional deficiency and infection.During severe burn shock,severe thermal injury can cause neutrophils,macrophages,monocytes,lymphocytes,and natural killer cells to release cytokines to participate in the body's defense response,and can also act on normal cells and tissues.Severe burns not only damage the function of the skin,but can even induce systemic inflammatory response syndrome(SIRS)and multiple organ failure(MOF)in the clinic.Whether or not to survive the severe burn shock phase smoothly is considered a critical period of treatment,but the underlying pathological mechanism of the severe burn shock phase is not clear.Exosomes are extracellular vesicles secreted and excreted by cells,which are lipid bilayers in nature.Most of the contents are proteins,miRNAs,and mRNAs.The materials play an important role in exchanging information between cells through transport.At present,more and more evidences show that miRNAs in exosomes can act on target genes to regulate the pathological processes of diseases related to diseases.Our research group studies the pathophysiological molecular mechanism of severe burn shock from the perspective of exosomes in serum.ObjectiveProve the existence of exosomes in the serum of rats after severe burns,and conduct relevant identification of morphological characteristics.Obtain differential miRNA expression profiles of exosomes in the 24h group and 48h group after severe burn.Bioinformatics analysis of miRNA target genes will deepen the understanding of the pathophysiological molecular mechanism of severe burn shock,explore whether exosomes have the potential to be a diagnostic marker of severe burns,and provide a new direction for molecular targeted therapy of diseases.Methods1.Establishment of animal model of severe burn rats and specimen collection:According to the principle of random grouping,two rats were divided into a severe burn experimental group and a sham burn group for exosome identification.In addition,18 rats were randomly divided into a sham burn group,and two experimental groups were used for exosomal sequencing:blank control group(sham group[n=6]);severe burn early shock group(after severe burns)24h group[n=6]);severe burn late shock group(48h group after severe burn[n=6]).The SD rats in the experimental group(male;200-220g;2 months old)were shaved with their backs immersed in a 97 ?thermostatic water tank and exposed for 15 seconds to establish a third-degree burn model.A false scald model was established in the constant temperature water tank for 1 5s,and the burned area accounted for 20%of the total area.Blood was collected from the ophthalmic vein of the rats,and blood was collected at different time points in different groups.After the blood collection,the serum was separated and stored frozen.2.Extraction and identification of exosomes:Use the kit to extract exosomes from all serum samples,and to identify exosomes in rat sera from the sham burn group and the severe burn test group,respectively.The existence of exosomes was verified by transmission electron microscopy(TEM),particle tracking analysis(NTA),and western blotting(WB).3.Second-generation sequencing of miRNAs in exosomes:First,total RNA of exosome weight was extracted,then miRNA library was constructed,and the quality of the library was tested.The expression profile of differentially expressed miRNAs obtained using the Illumina Hiseq2500 sequencing system was used to analyze differentially expressed miRNAs and predict target genes.4.Functional enrichment analysis of differentially expressed miRNA target genes:After obtaining differential miRNA expression profiles of each group,perform GO(GO Ontology,Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes,Kyoto)Encyclopedia of Genes and Genomes)Bioinformatics Analysis.5.Functional construction of protein interaction network:The most significant common differentially expressed miRNA target genes were analyzed by STRING database to construct a protein-protein interaction network(PPI)and visualized using Cytoscape.Use MCODE mode to trim and identify core modules from this network diagram.6.RT-PCR verification:select miRNAs that are differentially expressed in the experimental group for verification,and verify the accuracy of the results of the second-generation sequencing experiments.7.Statistical analysis method:A one-way analysis of variance was used to select a two-tailed range with a p value of<0.05.The differences in miRNA expression between the sham burn group,the severe burn group at 24 h and the severe burn group at 48 h were calculated.Results:1.Exosomes were successfully isolated from the sera of the severe burn experimental group and the sham bum group,and a typical double-concave dish shape was observed under a transmission electron microscope;the average particle size of the exosomes of the severe burn experimental group was 192.8 The average particle size of exosomes in the sham group was ± 65nm,and the average particle size of exosomes was 169.6± 59.2nm.The WB test in the severe burn group showed positive expressions of CD63,CD81,and HSP70,and the positive expressions of CD9,CD63,and CD81 in the sham group.2.Compared with the sham group,34 differentially expressed miRNAs were detected in the 24h group after severe burns,and 63 in the 48h group after severe burns.When P<0.005 and log2(foldchange)|?1,miRNA-339-5p,miRNA-206,miRNA-378d,miRNA-1,miRNA134-5p,and miRNA-365a-5p was significantly different from sham group.3.GO function enrichment analysis shows that target genes that differentially express miRNAs during severe burn shock are mainly related to cellular processes,metabolic processes,biological regulation,and the cellular immune system;KEGG pathway analysis shows natural killer cell-mediated cytotoxicity,B cells Receptor signaling pathway,axon guidance pathway,pyruvate metabolism pathway,long-term enhancement effect signaling pathway,type 2 diabetes pathway and tumor proteoglycan pathway were significantly enhanced.Conclusion:1.To prove the existence of exosomes in rat serum after severe burns,and to obtain the expression profile of exosome differential miRNAs during severe burn shock by sequencing and analyzing exosomes.2.The differentially expressed miRNAs of exosomes are different in the early and late stages of severe burn shock.3.Bioinformatics analysis of target genes that differentially express miRNAs after severe burns proves that the body has immune dysfunction after severe burns.4.MiRNA-206,miRNA-223-5p and miRNA-1 may be markers for early diagnosis of SIRS and MOF caused by severe burns.The let-7 family miRNA,miRNA-122,miRNA-21,miRNA-223 may be Related miRNAs that cause hyperglycemia after severe burns may become new therapeutic targets in the later stage.