| Objective: 1.To explore the construction of animal models with different outcomes for survival and death in sepsis.2.To find differentially expressed genes in two groups,the transcriptomes of the sepsis survival and death groups were sequenced by using high-throughput sequencing.3.In order to find the key genes that determine the different outcomes of sepsis survival and death,bioinformatics was used to analyze the differentially expressed genes,and the key genes were verified by RT-PCR to ensure the reliability of the results,which provided new targets for sepsis research and clinical diagnosis and treatment.Methods: 1.Mice were intraperitoneally injected with PBS and lipopolysaccharide solution at 0 mg/kg,10 mg/kg,20 mg/kg,30 mg/kg,40 mg/kg,50 mg/kg to construct sepsis models with different mortality,and there are 10 mice in each group.The temperature and activity indicators of the mice were observed to determine whether the sepsis models were successful.The observation was continued for 7 days,and the survival time of the mice was recorded to draw a survival curve.Groups with 30% and 85% mortality rates were defined as the most suitable models.2.The above-mentioned two different concentrations of lipopolysaccharide solutions were used to construct mouse models ofsurvival and death in sepsis,and an equal volume of PBS solution was used to construct a blank control group.Immediately after these two groups of peripheral blood samples were taken at appropriate time points,and the expression levels of plasma inflammatory factors TNF-α,IL-1βand IL-6 were measured by ELISA method.3.Peripheral blood samples from the survival and death groups of sepsis mice were collected for high-throughput sequencing of the transcriptome using the Illumina platform.At the same time,these measured original gene expression data are uploaded to the I-sanger cloud platform for screening of differentially expressed genes,The GO(Gene Ontology)and the KEGG(Kyoto Encyclopedia of Genes and Genomes)databases were used to perform GO functional annotation analysis,KEGG biological pathway analysis and KEGG metabolic pathway analysis on the differential genes that were screened,and the STRING database was used to build a protein interaction network module to find sepsis survival Key genes for death.4.RT-PCR technology was used to reverse the expression of the selected key genes in the sepsis survival group and the death group,and the relative expression of key genes was statistically analyzed using SPSS22.0 to determine the reliability of the difference.Results: 1.By observation,except for the mice were given PBS,the other mice showed various degrees of mental depression,unresponsiveness,and accelerated breathing.At 2 h,the mice at the dose of 50 mg / kg began to die,and over time,some mice began to havesymptoms such as eyelid redness,swelling,loosening of hair,fluffy hair,less luster,and decreased body temperature.During the 7-day observation period,asymptomatic and mildly symptomatic mice survived,severely symptomatic mice died,and the time of death was mostly concentrated in the first 48 hours.According to the administered doses are 0 mg/kg,10mg/kg,20 mg/kg,30 mg/kg,40 mg/kg,50 mg/kg,the mortality rates of mice is 0%,10%,30%,50%,90%,100%,it was determined that the optimal dose for the sepsis survival group was 20 mg/kg and the death group was 40 mg/kg.2.The expressions of inflammatory factors in the peripheral blood plasma of the survival group,the death group model and the blank control group were measured by ELISA method.The levels of inflammatory factors in the sepsis survival and death groups were significantly higher than those in the control group and the difference was statistically significant;The expression levels of inflammatory factors were also significantly different between the survival and death groups,and the differences were statistically significant.3.By analyzing the transcriptomics of the mouse models of sepsis survival and death groups,994 differential genes were screened out,in which 553 genes were up-regulated,441 genes were down-regulated,and the genes Gmpr,Shmt2,Prdx2,Orm1,Mfhas1,Aldh2,Gsr were identified as key genes related to sepsis death,and genes Gmpr,Shmt2,Prdx2,Orm1,Mfhas1 were positively correlated with death,while genes Aldh2,Gsr were negatively correlated with death.4.The RT-PCR method verified therelative expression of key genes in the sepsis production group and the death group.The results were in good agreement with the bioinformatics analysis,and the difference was statistically significant.Conclusions: 1.The animal models of the sepsis survival group and death group were successfully constructed by using different doses of lipopolysaccharide,and the optimal dose for the sepsis survival group was determined to be 20 mg/kg,and the best dose for the death group was40 mg/kg.2.By using high-throughput sequencing technology to perform peripheral blood transcriptomics sequencing of disease model mice and use bioinformatics software to analyze the sequencing data,it was speculated that the genes Gmpr,Shmt2,Prdx2,Orm1,Mfhas1,Aldh2,Gsr may be the key genes for different outcomes in sepsis.3.The detection of the relative expression of key genes has proved the feasibility and superiority of high-throughput sequencing technology and bioinformatics analysis in the study of sepsis,which providing new ideas for the study of sepsis,and also providing a new research target for the diagnosis and treatment of sepsis. |
PDF Full Text Request