| Research background and purpose:Severe sepsis is one of the major reasons for ICU admission.It can be caused by bacteria,toxins and viruses,among which Gram-negative bacteria and endotoxins produced by the Gram-negative bacteria are most intensively studied in severe sepsis.Clinical data have indicated that Gram-positive bacteria are also among the major pathogens causing severe sepsis in ICU.In recent years,the incidence and drug resistance of Staphylococcus aureus infection have been rising year by year.Therefore,looking for effective measures against the high prevalence and high mortality of Staphylococcus aureus infection in ICU has become a priority.The roles of Treg cells in CLP-related intraperitoneal infection or LPS-induced sepsis are generally recognized,but few studies are devoted to the relationship with infection by Gram-positive cocci and Staphylococcus aureus.Therefore,to clarify the regulatory roles of Treg cells in the onset of sepsis caused by Staphylococcus aureus and the mutual influence between the two will shed new light on the non-antibiotic treatment of sepsis caused by Staphylococcus aureus.We proposed the following assumptions: Treg cells work in the immunity against Staphylococcus aureus infection by regulating Teff cells.Reducing the excessive activation of immune response at the early stage of Staphylococcus aureus infection via the Treg cells and maintaining immune homeostasis will not only reduce the damage and mortality caused by sepsis,but also promote the action of Teff cells in removing the pathogens and reducing the overall mortality of Staphylococcus aureus infection.Therefore,we induced early systemic inflammatory response in animals using large doses of Staphylococcal enterotoxin B(SEB),which was followed by increasing or inhibiting the CD4+CD25+Treg cells.Thus the effect of Treg cells on SEB-induced early inflammatory response was observed and the potential signaling regulatory mechanism was analyzed.Part 1: The sepsis induced by staphylococcal enterotoxin B in rats and its observationObjective: Early inflammatory response caused by different doses of SEB was observed;the optimal dose at which severe inflammatory response was induced without causing damage to multiple organs or death of the animals was determined.Then the changes in the levels of inflammatory mediators in the peripheral blood were measured.The changes in the intensity of immune response were evaluated,thus laying the basis for further studies on pathogenesis and treatments.Methods: 1.Male SD rats were randomly divided into control group,low dose group,middle dose group and high dose group,each group of 10 rats.The rats were administered with 15 ug / 100 g,30ug / 100 g,45ug / 100 g SEB for 72 h,and the control group is injected the normal saline;2 Observe activity of the rats,the appearance of changes and 48 h survival rate after modeling,and record the general condition of the rats.Inflammatory factors were detected at 3h、6h、12h、24h、48h by ELISA.IFN-γ,TNF-α and IL-6 in each group was significantly higher than that in the control group,the difference was statistically significant(P <0.05).4.Each group of death or to life after 48 hours immediately after the death of lung,liver,kidney,microscopic observation form the result of the change.5.SPSS20.0 software was used for statistical analysis and measurement data were expressed by the mean ± standard deviation.Comparisons among groups were analyzed by one-way ANOVA;comparisons between two groups were tested by LSD method;survival rates were analyzed with Kaplan-Meier method and Log-rank test and P<0.05 indicated that the difference was statistically significant.Result1.The general situation and survival rate observed: 24 hours after modeling,rats activities were all normal in the control group and the low-dose group,and middle-high dose group showed the typical sepsis symptoms;48h after the control group,low,medium and high dose Group survival rates were 100%,100%,50%,20% respectively.2.The expression of TNF-α,IFN-γ and IL-6 in the control group were significantly lower than those in the low,middle and high dose groups at all time points after modeling.The inflammatory inflammation in the low,medium and high dose groups showed a progressive increase trend,reached a peak at 24 hours,followed by a downward trend.The expression of inflammatory factors was positively correlated with SEB dose.3.Observation of changes in important organs under the light microscope: No apparent changes were observed in the lung,liver and kidney under the light microscope for the control and low-dose groups.But in the moderate-and high-dose groups,infiltration by large amounts of inflammatory cells,bleeding and necrosis were observed in the rat lung,liver and kidney.ConclusionSystemic inflammatory response of desired intensity was induced by the administration of 30μ g/100 g SEB via the femoral vein without causing damage to multiple organs.The sepsis model in rats was thus established for the study of early-stage sepsis.The survival rate was 60% at 48 h,and the intensity of inflammatory response and the damage to multiple organs positively correlated to SEB dose.Effects of Treg activity on SEB induced sepsis in rat Objective:We studied the potential role of CD4+CD25+Treg cells in reversing the early inflammatory response caused by SEB via either increasing or inhibiting CD4+CD25+Treg cells in rats.The regulatory mechanism of CD4+CD25+Treg cells in this process was also discussed.1.Two rounds of sorting were performed using immunomagnetic beads to isolate high-purity and high-activity Treg cells from rat spleen for subsequent experiments.2.Treg cells reduced the damage to the organs and lowered the early mortality of rats with SEB-induced sepsis.The mechanism was related to the inhibition of pro-inflammatory factors such as TNF-α,IFN-γ and IL-6 by upregulating the Treg cells.This further led to the alleviation of early excessive inflammatory response caused by Staphylococcus aureus infection.In contrast,after blocking the Tregs with PC61,the resistance to SEB was decreased significantly in rats.The specific signaling mechanism remains to be investigated.The changes of TNF-α,IFN-γ and IL-2 were measured by ELISA.The expression of TNF-α,IFN-γ and IL-6 in the rats were measured at 6h,12 h,24h and 48 h after injection.The number of Treg cells in peripheral blood was detected by flow cytometry at each time point.(5)Statistical analysis was performed using SPSS 20.0 statistical software.The data were measured by the mean ± standard deviation.The comparison between the two groups was based on the independent sample t test.The comparison between the two groups was based on the one-way ANOVA.P <0.05 was statistically significant,P <0.01 was significantly different.ResultThe purified CD4 + CD25 + Treg cells were isolated from the spleen T lymphocytes by 2 immunomagnetic beads.The cell viability was maintained at 96-99% by trypan blue staining.The purity of the cells was 92.4%-98.2% by flow cytometry.2.the general situation and survival rate observed: 24 hours after modeling,SEB group and blocking group showed typical sepsis symptoms;Treg group early free exercise,compared with the other two groups,clinical symptoms semi-quantitative score significantly Lower than the other two groups(P <0.05).The survival rates of SEB group,blocking group and Treg group were 70%,50% and 90% respectively after 48 hours.(P <0.05).The postoperative SEB group increased continuously,which was significantly higher than the initial level of the model(P <0.01).There was significant difference between the three groups(P <0.05)Compared with SEB group,the inflammatory factors were higher in the blocking group than in the latter group(P <0.05 or P <0.01).Compared with SEB group,the inflammatory factors were lower in the Treg group than in the latter(P <0.05 or P <0.01).(P <0.05).The number of Tregs in the SEB group was significantly higher than that in the early stage(P <0.01).Compared with SEB group,the Treg ratio of the blocking group was lower than that of the latter.(P <0.05 or P <0.01).Compared with SEB group,the Treg ratio was higher in the Treg group than in the latter(P <0.05 or P <0.01).In conclusion1.The use of immunomagnetic beads two sorting separation of rat spleen can be high purity,good activity of regulatory T cells,suitable for further study.2.Treg cell activity in the staphylococcal enterotoxin B-induced sepsis model can reduce the degree of early organ damage and reduce early mortality,the mechanism is to upregulate the level of Treg cells can inhibit proinflammatory TNF-α,IFN-γ,IL-6 secretion,can reduce the excessive inflammation of Staphylococcus aureus infection,on the contrary,PC61 blocked or blocked Treg after rat tolerance to SEB significantly reduced levels.But its specific signal mechanism needs further study. |
PDF Full Text Request