| Sepsis,a serious complication of infection and trauma,which could rapidly progress to septic shock and multiple organ dysfunction,seriously threatens the life of the patient.The mortality rate was as high as 40 to 60%.The pathophysiological process of sepsis was very complicated.In recent years,studies have found that vascular endothelial injury was closely related to the development of sepsis,which was the primary link that affects the prognosis of sepsis.In the systemic inflammation stage of trauma or infection,it would cause vascular endothelial damage,which might increase the permeability of vascular endothelium,a large amount of fluid penetrate into the interstitial space,aggravate the hypoxia of tissue cells,induce the aggregation of proinflammatory mediators,and release a large amount of oxygen free radicals and Proteases,and further induce microcirculation dysfunction.Bone marrow-derived endothelial progenitor cells would be mobilized into peripheral blood,specifically differentiate into vascular endothelial cells,and repair damaged blood vessels.This repair was the only way for the body to repair damaged vascular endothelial cells.However,the signaling pathway and mechanism of its mobilization and activation were not yet clear.Recent studies have reported that changes in the expression level of long non-coding RNA in sepsis might participate in regulating angiogenesis of endothelial cells,which might indicate the prognosis of sepsis,and long non-coding RNA-NEAT2 could regulate vascular endothelial cells under hypoxic environment.Proliferation and migration function.In summary,we speculated that in sepsis,long non-coding RNA-NEAT2 might affect vascular endothelial cells by regulating the number and function of endothelial progenitor cells,which in turn affected the prognosis of sepsis.This study was to construct a mouse sepsis model,observe the changes in the number and function of endothelial progenitor cells during the progression of sepsis,detect the changes in the expression level of long non-coding RNA-NEAT2 in sepsis,and analyze the correlation between the number and function of endothelial progenitor cells and then to provide theoretical basis and experimental basis for exploring new targets for clinical treatment of sepsis.Part Ⅰ Changes and significance in the number and function of mouse endothelial progenitor cells during sepsisObjective:To establish a stable mouse sepsis model,isolate and culture mice bone marrow-derived endothelial progenitor cells,and provide experimental basis for studying the expression level of long non-coding RNA-NEAT2 and the number,proliferation and differentiation function of endothelial progenitor cells during sepsis progression.To detect the changes in the number of circulating endothelial progenitor cells and the function of bone marrow-derived endothelial progenitor cells during the progression of sepsis.Method:Thirty-six SPF-grade Balb/C mice(male,6-8 weeks,weight 27±2 g)were randomly divided into two groups:18 in experimental group,cecal ligation and puncture were performed and 18 in the control group.The changes of status,tumor necrosis factor-α,interleukin-6,vascular endothelial growth factor and liver and kidney function of the two groups of mice were observed at fixed time points.After the mice were sacrificed,bone marrow flushing fluid was obtained,and endothelial progenitor cells were isolated and cultured by differential adherence method,and identified by immunofluorescence staining,flow cytometric identification and double phagocytosis experiment.Peripheral blood was taken from the tail vein at fixed time points during sepsis,and the number of endothelial progenitor cells in the peripheral blood were detected by flow cytometry.Bone marrow-derived endothelial progenitor cells were isolated and cultured,and the angiogenic and migratory functions of endothelial progenitor cells of endothelial progenitor cells were detected at fixed time points.Result:After cecal ligation and puncture,the mice in the experimental group were depressed in mental state,and the plasma levels of tumor necrosis factor-α,interleukin-6,vascular endothelial growth factor,glutamic-pyruvic transaminase,glutamic-oxalacetic transaminase and creatinine were significantly changed,which were significantly higher than those in the control group.Bone marrow mononuclear cells were isolated and cultured by differential adherence method,and identified as endothelial progenitor cells by immunofluorescence staining(CD31+,CD34+++),flow cytometric identification(CD31~+23.78±4.12%,CD34~+90.09±3.89%,CD133~+85.52±2.28%,CD309~+74.92±3.32%)and double phagocytosis test(double positive>80%).During the progression of sepsis,the number of endothelial progenitor cells in peripheral blood increased significantly,the angiogenesis and migration function of endothelial progenitor cells increased significantly.Conclusion:A stable mice sepsis model was established by cecal ligation and puncture method successfully,and endothelial progenitor cells were isolated and cultured.Part Ⅱ Changes in endothelial progenitor cell function and expression of long non-coding RNA-NEAT2 during sepsisObjective:To explore the correlation between the number and function of endothelial progenitor cells and long non-coding RNA-NEAT2.Method:Bone marrow-derived endothelial progenitor cells were isolated and cultured,and the expression levels of long non-coding RNA-NEAT2 of endothelial progenitor cells were detected at fixed time points.Down-regulate the expression of long-chain non-coding RNA-NEAT2 and observe the functional changes of endothelial progenitor cells.Result:Long non-coding RNA-NEAT2 expressed in the cells of the endothelial group of the experimental group gradually increased,and the highest abundance was 2.229±0.175,twice the normal level;while the control group was expressed in mouse EPCs cells after sham operation The abundance of long non-coding RNA-NEAT2 was comparable to that before modeling,and no significant increase was observed.The difference in abundance of long non-coding RNA-NEAT2 between the two groups of mice was statistically significant(p<0.05).After correlation analysis,the expression of lnc RNA-NEAT2 was positively correlated with the number of peripheral blood EPCs,angiogenesis function and migration function of EPCs.The Pearson coefficients were 0.935,0.842,0.849,p<0.05.After down-regulating the expression of long-chain non-coding RNA-NEAT2,endothelial progenitor cell angiogenesis function and migration function decreased.Down-regulate the expression of long-chain non-coding RNA-NEAT2 and observe the functional changes of endothelial progenitor cells.Conclusion:In sepsis mouse models,long non-coding RNA-NEAT2 was associated with the number and function of endothelial progenitor cells.Down-regulate the expression of long-chain non-coding RNA-NEAT2 and observe the functional changes of endothelial progenitor cells. |
